Major and Minor Groove Conformations of DNA ... - ACS Publications

Aug 9, 1994 - than 10 kcal/mol more stable than the major groove structurefor all sequences. As a result, minor groove adducts provided most of the...
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Chem. Res. Toxicol. 1995,8,117-127

117

Major and Minor Groove Conformations of DNA Trimers Modified on Guanine or Adenine by 4-Aminobiphenyl: Adenine Adducts Favor the Minor Groove Robert Shapiro,*9? Stephen Ellis,? Brian E. Hingerty,$ and Suse Broydes Chemistry and Biology Departments, New York University, New York, New York 10003,and Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received August 9,1994@

We have studied the conformational effects of 4-aminobiphenyl modification at C-8 of guanine or adenine on double-stranded DNA trimers. We used sequences with the modified purine at the central base pair and all 16 possible neighboring sequences a t the outer pairs. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to survey the conformation space of these adducts, using a total of 1280 starting structures both in the modified guanine series and in the modified adenine series. Conformer families in which the bound 4-aminobiphenyl was located in the DNA major groove, and in the minor groove, were located for both adenine and guanine modification. In the modified guanine series, the major and minor groove families were roughly comparable in energy, and the sequence context determined which was more stable in a particular case. In the modified adenine series, however, the minor groove structure was more than 10 kcaVmol more stable than the major groove structure for all sequences. As a result, minor groove adducts provided most of the global minima in the adenine-modified series. This result may be relevant to a previous mutagenesis study [Lasko et al. (1988) J. Biol. Chem. 263, 15429-154351 in which the hot spot of most frequent occurrence was located at a n adenine, in the sequence GAT.

Introduction “Incontrovertible”epidemiological studies have implicated 4-aminobiphenyl (ABP)l as a cause of human bladder cancer. Although its industrial use has been suspended, human exposure continues through cigarette smoke and various environmental sources (reviewed in ref 1). Chronic exposure to this amine produces liver and bladder cancer in rats, and tumorigenesis correlates well with the accumulation of the principal adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl(I, Figure 1)in DNA (2). It has been suggested that this adduct plays the major role in causing the bacterial mutagenesis observed with activated derivatives of ABP (3, 4). The biological importance of this and related adducts has stimulated investigations of their effect on DNA structure. In a recent NMR study of a 15-mer DNA duplex containing the guanine adduct (I), a major conformer and a minor conformer were observed (5). The major conformer had a normal B-DNA structure, with paired bases and the biphenyl residue in the major groove. The minor conformer was not identified, but intercalated or minor groove positions for the bound aminobiphenyl were suggested. A major groove position for the aminobiphenyl-guanine adduct in DNA had been predicted earlier on the basis of computations in one sequence (6). In the case of the related amine, 2-aminofluorene, a conformation with the amine in the minor groove has been established by NMR and energy-minimization studies on a modified DNA 11-mer,with adenine opposite the adduct (7).More recent studies of DNA duplexes con~~~~~

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Department, New York University. Oak Ridge National Laboratory. P Biology Department, New York University. Abstract published in Advance ACS Abstracts, December 1,1994. Abbreviatipn: ABP,4-minobiphenyl.

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taining 2-aminofluorene in different sequences, with the usual partner C opposite modified G, have suggested a mixture of conformers. As in the case of biphenyl modification, one of them had a normal B-DNA structure, with paired bases and the aminofluorene residue in the major groove. The other was felt to be in a more shielded position, either in the minor groove (8)or inserted into the helix (8,9).These results have suggested that DNA sequence may play an important role in determining the conformation of a bound amine. In addition to the adduct at guanine, however, a smaller amount of an adenine product, N4deoxyadenosin8-yl)-4-aminobiphenyl(11, Figure 11, is formed in the reaction of activated ABP derivatives with DNA (reviewed in ref 11, and it has been suggested that this adduct plays a role in the transformation of human uroepithelial cells in culture by aminobiphenyl metabolites (10, I1 1. An A-T pair represents a hot spot for ABP mutagenesis in Escherichia coli virus M13mplO (3). In another system, it has been suggested that N-(deoxyadenosin-8-yl)-Z-aminopyrene is the lesion responsible for 2-nitropyrene mutagenesis in Salmonella typhimurium strains TA96 and TA104, which have A-T base pairs as mutational hot spots (12).An adenine adduct of l-aminopyrene was earlier suggested as the cause of the small proportion of base substitution and frameshift mutations at A-T base pairs observed in E. coli uvr- lysogens treated with 1-nitrosopyrene(13).More broadly, the speculation has been made that “arylamine adducts with dA,though quantitatively rare, may be of particular significance to mutagenesis and possibly carcinogenesis’’(14). No experimental studies have been performed, however, on the conformation of single- or double-stranded DNA containingABP or related amines bound to adenine. In this work we have used our molecular mechanics

0893-228x/95/2708-0117$09.00/0 0 1995 American Chemical Society

118 Chem. Res. Toxicol., Vol. 8, No. 1, 1995

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I

It

Figure 1. Guanine (I) and adenine (II)adducts of 4-aminobiphenyl. R = H. The dihedral angle designations (A-B-C-D) for the aminobiphenyl to base linkage are as follows: a’: NS-C&amine N-C4(biphenyl); p’: C8-amine N-C4(biphenyl)-C5 (biphenyl); y’: C6-Cl-Cl’-C6. The angle A-B-C-D is measured by a clockwise rotation of D with respect to A, looking down the B-C bond. The central bond for the dihedral angles a’, /3’, and y’ are labeled in I. The numbering system of the biphenyl system is given in 11.

The dihedral angle designations for the sugar backbone are as follows: a: 03’-P-O5’-C5‘; p: P-O5’-C5’-C4‘; y : 05’-C5‘-C4‘CY; E : C4‘-CY-OY-P; 5;: C3’-03’-P-05’; x (pyrimidines): 04‘-Cl’-Nl-C2; x (purines): 04‘-Cl’-N9-C4. Sugar pucker is defined by the pseudorotation parameter, P (25).

program, DUPLEX, to compare the effects of the adenine and guanine adducts of ABP on the conformation of double-stranded DNA trimers. This was done for all 16 possible nearest-neighbor sequence contexts around a central adenine or guanine adduct. A strategy was employed that specifically searched for major and minor groove adducts. Using the trimer results, we have modeled duplex nonamer conformations that illustrate the minor and major groove conformations for the adenine and guanine adducts. We have found a striking difference between the adenine and guanine series which may be relevant to their mutagenesis.

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Methods Minimized potential energy calculations were carried out with DUPLEX, our molecular mechanics program for carcinogenmodified nucleic acids (15).DUPLEX performs potential energy minimizations in the reduced variable domain of torsion angle space. It employs a force field for nucleic acids that was devised in the laboratory of Olson (16,17). A hydrogen bond penalty function (15,18)is employed in all first stage minimizations to aid in the location of any type of designated hydrogen-bonded structure, or a denatured site when the function is not employed at a particular base pair. This penalty function makes no contribution to the energy when a selected hydrogen-bonding scheme has been achieved, but penalizes the energy when the selected pattern is not found. Thus, the function guides the minimization algorithm toward structures with the chosen hydrogen bonds. It is released in a subsequent minimization so that final computed structures are unrestrained minimum energy conformations. In the present work, the adjustable weight in this penalty function was assigned a value of 7.5 kcaY (mol&) in the trimer studies and 30-50 kcal/(mol*A2)in the nonamer work. The hydrogen bond penalty function was employed to search for five different hydrogen-bonding possibilities a t the modified base pair. Three of them, Watson-Crick, Hoogsteen, and “wedge”(18), are illustrated in Figure 2 for guanine and Figure 3 for adenine. The other two involved modified syn guanine (or adenine) opposite anti cytosine (or thymine) and modified anti guanine (or adenine) opposite anti cytosine (or thymine), with no hydrogen bonding sought. The hydrogen bond penalty function was also employed to locate standard Watson-Crick base pairs at the other sites. Torsion angles for the DNA starting conformation were those of the B-DNA fiber diffraction model (191,except that syn purines a t the lesion site were oriented with glycosidic torsion angles of 60”. Torsion angle definitions are given in Figure 1. Sixteen orientations of the carcinogen-base linkage torsion angles were employed as starting conformations for the energy minimizations of each hydrogen-bondingtype: a’ = O”, go”, 180”, 270” in combination with /3’ = O”, go”, 180”, 270” . This combination of 16 a’/3’ values, together with 16 sequences and 5 different hydrogen-bonding possibilities required a total of 1280 starting structures for each type of adduct. Following these first stage trials, a second minimization was performed for each resulting structure without the hydrogen bond penalty function. Those structures that employed protonated bases were

0-

R W d g c G(syn)-C(anti) Denatured Structures: G(anti) C(anti) G(syn) C(anti)

Figure 2. Guanine-cytosine hydrogen-bonding schemes. R = the remainder of the polynucleotide chain.

R

Watson Crick: A(mti)-T(8nti) 0

Wedge A(syn)-T(anti) h a t u r d Structure: A(8nti) T(anti) A(syn) T(anti)

Figure 3. Adenine-thymine hydrogen-bonding schemes. R = the remainder of the polynucleotide chain. deprotonated and minimized again in a terminal step, a s in earlier work (18). This procedure was necessary because energies of protonated and unprotonated forms cannot be compared: they contain different numbers of atoms. The geometry of the carcinogen and the linkage site, and the force field parameters (torsional potentials and rotation barriers), were the same for the guanine adduct as those used in earlier

DNA Trimers Modified by 4-Aminobiphenyl work (20). The same geometry and parameters were also used for the adenine adduct, except that revised partial charges were assigned at N7, CS, and N9 (-0.1137, 0.3507,and -0.3563, respectively). Bases and sugars in trimers are referred to numerically according to the following scheme: 123.456,in which 1 and 4 are the 5’residues of the two chains and 2 is the modified base. “Base pair” 1 refers t o the 1-6 pair and “base pair 3” to the 3-4 pair. In some cases an additional trial was run for a particular sequence using the angles of a favored minimum that had already been located in another sequence. If a particular hydrogen-bonding scheme had been sought for the existing minimum, this was employed in the new search. This procedure was employed to maximize the possibility that the lowest energy variant of a given structural type had been located, in view of the multiple minimum problem. Nonamers were generated from selected trimers by embedding the trimer in a B-DNA (19) nonamer of the desired sequence and minimizing the energy in stages as described above. Thus, the starting torsional angles were those of the trimer, flanked by two B-DNA trimers, to constitute a nonamer.

Results Initially, global minima and others within 2 kcal/mol of the global were examined for all sequences and classified into three types: (1) Major groove adducts: This class included conformers where modified guanine or adenine was anti and retained some bonding with its partner on the normal strand. The neighboring bases were fully paired, and the torsional angles were generally in the range associated with B-DNA. The biphenyl group was positioned in the major groove of the helix. Generally, these were produced from trials with anti guanine and Watson-Crick hydrogen bonding. (2) Minor groove adducts: The modified guanine or adenine was syn, and the neighboring base pairs were opposite each other and retained some hydrogen bonding, even though they might be bent out of coplanarity. Thus the overall structure approximated a double helix with the biphenyl in the minor groove. These structures were generally produced from trials with syn guanine and Hoogsteen or wedge hydrogen bonding. The feasibility of both syn and anti conformations for aminobiphenyl-modified deoxyguanosine and the twisted nature of the biphenyl residue (observed in all conformers) had been delineated in earlier conformational searches with a modified deoxydinucleoside monophosphate (20). (3) Rearranged structures: The term was applied to a variety of structures which no longer resembled a double helix, with the modified purine syn or anti. The hydrogen bonding at one or both outer base pairs was broken, and one or more of these bases had moved out of helical position, in most cases to a position beyond the plane of the adjacent central base. Each of these types is discussed below, for guanine and adenine modification. An attempt was also made to build a representative of each type to a duplex nonamer, to examine its characteristics and stability in a larger duplex. The nonamers are also discussed in separate sections below. Guanine-SubstitutedMajor Groove Adducts. Trimer Sequences. The torsional angles of the global minima for the series N W W are given in Table 1. A structure with anti guanine in a normal position in a DNA helix and aminobiphenyl in the major groove furnished the global minimum in 7 sequences (see Table 2). We also located a very similar structure for the remaining sequences, and the energy of these conformers

Chem. Res. Toxicol., Vol. 8, No. 1, 1995 119 with respect to the global minimum for that sequence is also given in Table 2. The 16 structures differed by less than 20” from one another in their internal torsional and pseudorotation angles, and, with one exception, from the angles of the corresponding series of unmodified B-DNA trimers. The exception was in the pseudorotation angle of modified guanine, which varied from 104”to 122”(04’endo) in the biphenyl-modified trimers and from 157”to 171” (C2’-endo) for the unmodified trimers. (We have found that the values of the terminal angles, j31, y1, and c3, as well as those of the thymine methyls have litttle effect on the overall shape of a conformation.) This conformer type is illustrated for GpCPPpG.CpCpC (Figure 4a). The a‘ value of 168” sets the amine bonds in a position almost coplanar with the guanine ring. The proximal biphenyl ring stacks on the 2’-CHz protons of the 5’-neighborin deoxyribose, and the amine NH has a stretched (2.41 ) H-bond to 0-3’ of that sugar. H-2 of aminobiphenyl is positioned above the imidazole ring of the 5’-neighboring guanine. The distal ring of biphenyl is exposed. The general features of the major groove adduct trimer family (and the derived nonamer, see below) agreed with those deduced from an NMR study of an aminobiphenyl modified 15-mer (5)and our earlier computed structure (6). The major conformer in that study contained an intact B-DNA helix, with the H-bonds from modified anti G to C intact; the biphenyl residue was positioned in the major groove and was free to rotate, but had some interaction with the 5’-neighboring nucleotide. Although the 16 members of our major groove trimer family are similar in structure, they vary considerably in energy, and this variance plays an important role in determining whether a particular conformer represents the global minimum in its sequence. An energy comparison is possible in sequences which have the same number of GC and AT pairs, as they are isomers. For example, relative energies in kcal/mol for the major groove duplex trimer conformers with all G.c pairs are as follows (only modified strand sequence given): WpC, 0.0; G W P G , 2.6; C W p C , 4.1; and CGABPG, 6.6. Thus the sequence PuGABpPy was most stable and PyGABpPu least stable. This same order applied in other isomeric series as well, with sufficient numerical consistency to permit rules to be drawn up. Thus in comparison to the most stable structure, PuCPpPy, we find the following:

1

energy of PuGABpPu: 2.4-3.7 kcaVmol

energy of PyGABpPy: 3.5-4.1 kcal/mol energy of PyGABpPu: 6.1-8.2 kcaVmol This relationship was not caused by the aminobiphenyl as it also held (with slightly more numerical variability) for the unmodified trimers. In the latter case, we are comparing the energies of PuPuPyPuPyPy (most stable) vs PyPuPwPyPuPu (least stable). The two sequence types, in terms of nearest-neighbor interactions, differ in the presence of 2 PuPy interactions in the former structure vs 2 PyPu interactions in the latter. In a recent evaluation of nearest-neighbor interactions in doublestranded DNA, their stability was found to be dependent upon ionic strength, with P u w P u P y more stable than PyPuPyPu for ionic strengths of 55 mM and higher (measured to 115 mM) (21). Our calculations using DUPLEX in this work did not employ explicit counter-

120 Chem. Res. Toxicol., Vol. 8, No. 1, 1995

Shapiro et al.

Table 1. Global and Other Significant Minima for Guanine-Modified Trimers and NonameP-d sequence d(GpG*pG) d(CpCpC) d(GpG*pA) d(TpCpC) d(GpG*pC) d(GpCpC) d(GpG*pT) d(ApCpC) d(ApG*pG) d(CpCpT) d(ApG*pA) d(TpCpT) d(ApG*pC) d(GpCpT) d(ApG*pT) d(ApCpT) d(CpG*pG) d(CpCpG) d(CpG*pA) d(TpCpG) d(CpG*pC) d(GpCpG) d(CpG*pT) d(ApCpG) d(TpG*pG) d(CpCpA) d(TpG*pA) d(TpCpA) d(TpG*pC) d(GpCpA) d(TpG*pT) d(ApCpA) d(GpG*pT)-9 d(ApCpC)-9 d(GpG*pT)-9 d(ApCpC)8

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€3 class 187 major 187 187 major 187 188 major 187 188 major 187 185 minor-ld 186 185 rearr 186 187 major 187 187 major 187 185 rearr 184 185 rearr 185 188 major 187 185 minor-bc 185 185 minor-u 185 185 rearr 185 186 minor-3d 185 185 minor-3d 186 181 major 186 181 minor 183

a All minima are global, except for the minor groove nonamer whose LW is 3.7 k d m o l . Torsional angles and pseudorotation parameters are in degrees. b The symbol G* represents guanine modified at the 8-position by 4-aminobiphenyl. Angles for the unmodified strand are given just below those for the modified strand. The residues on each strand are numbered from the %-end. The structure marked by the suffix ”9” represents the central trimer of a larger nonamer. Residue 1in the table then represents the fourth residue from the 5’-end of the nonamer chain.

Table 2. Classification and Stability (kcdmol) of Guanine-Modified Duplex Trimers modified sequence

minor major groove groove (type)

0.0 0.0 0.0 0.0 1.9 4.1 0.0 0.0 a

2.3 (Id) 3.5 (u) 5.0 (u) 3.8 (u) 0.0 (Id) 3.8 (Id) 1.3 (u) 0.2 (u)

modified sequence d(CpWPpGP d(CpGABPpAP d(CpGABPpC) d(CpGABPpT) d(TpwPpG) d(TpWPpAP d(TpWPpC) d(TpGABPpT)

minor major groove groove (type) 1.6 2.1 (u) 11.1 15.1 (u) 0.0 2.2 (u) 2.0 0.0 (bc) 0.0 (u) 2.8 7.0 (3d) 15.4 1.4 0.0 (3d) 0.0(3d) 4.6

In cases where neither major nor minor groove structure is

at 0.0 kcal/mol, then the global minimum is of the rearranged “stack of 4” type. (See the Rearranged Trimers section.)

ions, but rather simulated the screening effect of ions by using a distance-dependent dielectric function with a Debye screening parameter corresponding to a monovalent salt concentration of 0.1 M (22). Thus our results with unmodified trimers reflect the nearest-neighbor effects a t 0.1 M salt, and the stability of the modified major groove trimers follows those of the unmodified set. Guanine-SubstitutedW o r Groove Adducb. Nonamer Sequence. The major groove conformer of sequence GpGABPpT was embedded (see Methods) in a B-DNA nonamer of sequence GpGpGpGpWppTpGpTpG CpApCpApCpCpCpCpC and energy minimized, for comparison of energies with the minor groove conformer of

the same sequence. The major groove conformer was more stable by 3.7kcdmol. (The difference at the trimer level had been 3.8 kcallmol.) The angles of the central trimer of the nonamer are given in Table 1. The nonamer displayed the same features (Figure 5) in the vicinity of the modified G as those exhibited by the original trimer. The hydrogen bonds from modified G to its partner were intact and of normal length. The sequence employed for the guanine nonamers was the same as the mutagenic hot spot used for the adenine series, save that the modified adenine-thymine pair was replaced by a modified guanine-cytosine pair.

Adenine-Substitutedm o r Groove Adducts. Trimer Sequences. The torsional angles of the global minima for the series N A ? N are given in Table 3. A structure with anti adenine in a DNA helix and aminobiphenyl in the major groove could be identified in all 16 sequences. Unlike the case of the guanine adducts, the adenine major groove adducts provided no global minima and were less stable than the minor groove adducts in all sequences by 11.2-19.8 kcdmol (see Table 4). The torsional angles for a typical major groove adduct, GAABPT.ATC(illustrated in Figure 4b), have been included in Table 3. The internal torsional angles of the 16 adenine-modified major groove structures differed by less than 20”from one another and, with one exception, from the angles of the corresponding series of unmodified

DNA Trimers Modified by 4-Aminobiphenyl

Chem. Res. Toxicol., Vol. 8, No. 1, 1995 121

Figure 4. Stereoviews: (a) GpGABPpGCpCpC: Major groove adduct. (b) GpAABppT.ApTpC:Major groove adduct. (c) @GABppAJI'pCpA Rearranged "stack of 4" adduct. These stereoviews are prepared for use with a stereoviewer.To view with crossed eyes, the left and right images must be interchanged.

Figure 5. Stereoviews of GpGpGpGpGABPpTpGpTpGpApCpApCpCpCpCpC:Major groove adduct. See the caption to Figure 4.

B-DNA trimers. The exception was in the pseudorotation angle of modified adenine, which varied from 80" to 116" (04'-endo) in the adenine biphenyl-modified trimers and from 154" to 162" (C2'-endo) for the unmodified trimers. (In the guanine series, the pseudorotation angles had varied from 104" to 122" in the biphenyl-modified trimers and from 157" to 171" for the unmodified trimers.) Other differences between the major groove modified guanine and adenine conformer series were the following (average difference given in degrees): pseudorotation of modified residue (up to 30"), pseudorotation of partner of modified residue (E"), and C4 (15"). The position of the biphenyl residue in the major groove of the adenine-modified series is the same as in the guanine-substituted series, with the proximal ring stacking on H-2' and H-2" of the 5'-neighboring sugar and the distal ring exposed. There is a significant difference in the helix, however. The modified adenine to thymine base pair has a propeller twist of 23" in the structure shown in Figure 6 (this varies from 18" to 25" in other sequences). In addition, the A-amino to Tcarbonyl H-bond is broken (2.53 A), though the other

H-bond is intact. The plane of the base 3' to the modification remains parallel to that of modified adenine, while the other bases remain parallel to partner thymine. Attempts were made to place an adenine-modified trimer adduct into the conformation of the corresponding modified guanine trimers. On minimization, however, the adenine-modified trimers reverted to the propellertwisted state. This structure appears to reflect the necessary geometry of the adenine system, rather than to have arisen as an artifact of the search pathways. The same variation of energies among isomeric major groove structures with sequence that was observed in the guanine adduct series was also present for the adenine adducts. For example, relative energies in kcal/mol for the major groove duplex trimer conformers with flanking GC pairs are as follows (only modified strand sequence given): GAABpC,0.0; GAABPG,3.5; CAABpC,5.2; and CAABPG,8.6. Thus, as in the G-adduct series, the sequence PuAABpPywas most stable and PyAABPPUleast stable. This same order applied to the isomers with other flanking base pairs as well, with sufficient numerical consistency to again permit rules to be drawn up. Thus in comparison to the most stable structure, PuGABpPy,

122 Chem. Res. Toxicol., Vol. 8,No. 1, 1995

Shapiro et al.

Table 3. Global and Other Significant Minima for Adenine-ModifiedTrimers and NonameP-d sequence 81 d(GpA*pG) 179 d(CpTpC) 183 d(GpA*pA) 179 d(TpTpC) 179 d(GpA*pC) 184 d(GpTpC) 180 d(GpA*pT) 293 d(ApTpC) 184 d(ApA*pG) 180 d(CpTpT) 183 d(ApA*pA) 179 d(TpTpT) 183 d(ApA*pC) 184 d(GpTpT) 179 d(ApA*pT) 293 d(ApTpT) 184 d(CpA*pG) 184 d(CpTpG) 184 d(CpA*pA) 175 d(TpTpG) 183 d(CpA*pC) 184 d(GpTpG) 179 d(CpA*pT) 184 d(ApTpG) 184 d(TpA*pG) 180 180 d(CpTpA) d(TpA*pA) 183 d(TpTpA) 186 d(TpA*pC) 184 d(GpTpA) 179 d(TpA*pT) 180 d(ApTpA) 180 d(GpA*pT)” 179 d(ApTpC) 180 d(GpA*pT)-9 172 d(ApTpC)-9 190 d(GpA*pT)-9 178 d(ApTpC)-9 179 (I

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226 157 33 189 271 188 253 219 151 33 187 268 180 265 210 146 27 186 267 186 265 210 145 31 183 268 190 277 213 153 22 186 272 188 270 219 154 26 187 269 184 271 211 146 27 186 268 186 277 214 144 27 182 268 191 275 209 146 29 186 270 193 253 186 32 34 192 276 197 282 207 146 30 187 269 185 261 206 147 29 183 267 188 263 203 147 31 183 268 188 258 156 58 44 187 271 195 283 203 146 32 184 266 182 267 205 147 32 179 267 188 274 177 36 37 177 256 185 244 206 156 18 180 268 192 270 189 38 38 174 252 187 234

a3

296 284 301 295 301 287 304 292 302 295 304 296 298 294 302 294 304 293 299 286 299 289 302 287 303 291 300 287 302 292 302 292 308 293 298 293 312 303

83

y3

169 59 177 63 169 55 174 62 165 60 168 73 173 48 178 64 173 47 170 67 168 53 170 66 171 53 176 65 175 50 178 63 171 48 176 59 169 54 180 59 167 55 182 62 173 50 183 62 170 52 178 63 165 63 182 55 167 54 182 64 173 54 184 61 174 52 182 53 172 58 158 80 176 51 176 51

P3

x3

156 152 154 155 145 155 124 154 143 156 151 149 132 150 127 158 137 173 162 177 135 173 122 171 140 174 161 178 122 174 129 171 137 157 140 161 152 165

259 245 255 251 245 242 235 242 244 246 253 244 240 241 236 244 242 253 265 290 243 254 235 250 241 258 262 298 235 258 236 258 233 237 242 247 238 241

Me3

311 307 69 309 313 308

71

61 59 280 37

€3 class 186 minor 187 187 minor 186 187 minor 186 188 minor 186 187 minor 186 187 minor 185 188 minor 187 188 minor 186 188 minor 185 185 rearr 185 188 minor 185 188 minor 185 188 minor 186 186 ream 185 188 minor 185 188 minor 187 187 major 186 176 minor 176 183 major 184

All minima are global except for the major groove for d(GpA*pT), for which AE = 11.2 kcaYmol, and the major groove nonamer whose

AE is 8.9 kcavmol. Torsional angles and pseudorotation parameters are in degrees. The symbol A* represents adenine modified at the 8-position by 4-aminobiphenyl. Angles for the unmodified strand are given just below those for the modified strand. The residues on each strand are numbered from the 5’-end. The structure marked by the suffur “9” represents the central trimer of a larger nonamer. Residue 1 in the table then represents the fourth residue from the 5‘-end of the nonamer chain.

Table 4. Classification and Stability (kcdmol) of Adenine-ModifiedDuplex Trimers modified major minor sequence groove groove d(GpAABPpWb 13.7 0.0 0.0 d(GpAABPpA) 13.7 0.0 d(GpAABPpC) 11.3 d(GpAABPpT) 11.2 0.0 d(ApAABPpG) 15.0 0.0 0.0 d(ApAABPpA) 14.2 0.0 d(ApAABPpC) 12.0 0.0 d(ApAABPpT) 13.2 a

modified sequence

major minor groove groove 19.8 0.0 20.6 0.9 0.0 16.3 14.5 0.0 19.3 0.0 21.0 2.7 15.5 0.0 15.0 0.0

In cases where neither major nor minor groove structure is

at 0.0 kcaYmol, then the global minimum is of the rearranged “stack of 4” type. (See the Rearranged Trimers section.) In this sequence, the “A-minor” structure appeared at 0.1 kcaYmo1. The global minimum was a more distorted minor groove structure with base pair 1 showing buckling and propeller twisting.

we find the following: energy of PuAmpPu: 2.8-4.2 kcaVmol

energy of PyAABpPy:4.9-5.8 kcal/mol energy of PyAmpPu: 8.2-9.1 kcaVmol

As in the guanine series, the same approximate stability relations were also present in the unmodified trimers

and, therefore, were not caused by the presence of the carcinogen, but reflected nearest-neighbor base effects. Adenine-Substitutedm o r Groove Adducts. Nonamer Sequence. The major groove conformer of sequence GAmT was embedded (see Methods) in a B-DNA nonamer of sequence GpGpGpGpAABPpTpGpTpGCpApCpApTpCpCpCpC and energy minimized, for comparison of energies with the minor groove conformer built up by similar means in the same sequence. The torsional angles of the central base pairs of the nonamer are included in Table 4. This nonamer was 13.2 kcal/mol less stable than the minor groove structure (they had differed by 11.2 kcaymol, a t the trimer level). It displayed the same features (Figure 6) in the vicinity of the modified adenine as has the original trimer. The position of the biphenyl residue was the same. The propeller twist of the central modified base pair was 25”, and the distance from adenine amino NH to T carbonyl 0 4 was 2.72 A. Base pairs 3‘ to modified adenine were parallel to that adenine and those 5‘ to modified adenine were parallel to partner thymine. Guanine-Substituted Minor Groove Adducts. Trimer Sequences. In the guanine-substituted series, a number of low energy conformers had syn guanine, with the bases in approximate double-helical order and biphenyl in the minor groove of the helix (see Table 1).The modified guanine did not hydrogen-bond to the cytosine

DNA Trimers Modified by 4-Aminobiphenyl

Chem. Res. Toxicol., Vol. 8, No. 1, 1995 123

Figure 6. Stereoviews of GpGpGpGpAABPpTpGpTpGpApCpApTpCpCpCpC:Major groove adduct. See the caption to Figure 4.

Figure 7. Stereoviews: (a)TpcpBPpGpCpA Minor-u adduct. (b) ApGABPpWpCpT Minor-ld adduct. (c) TpWPpT-ApCpA Minor-3d adduct. (d) CpGABPpT*ApCpG Minor-bc adduct. (e) GpAABppCGpTpC: Minor groove adduct. See the caption to

Figure 4.

opposite it. The value of the torsional angle a’ was greater than NO”, which served to orient biphenyl in a 3’-direction (with reference to the modified strand) in the minor groove. In five sequences, a structure with these characteristics was the global minimum (see Table 2). Unlike the situation in the adenine adduct series (see below), these minor groove structures differed somewhat from one another and could be grouped into four differing subclasses. Each was represented by at least one global minimum (see Table 1for torsional angles). The types are listed below: (1)Relatively undistorted helix (“minor-u”): illustrated by TpCPPpGCpCpA (Figure 7a). In this series, the biphenyl residue has fair cross-strand contacts but weak contacts to its own strand.

(2) Base pair 1 distorted (the bases at an angle with hydrogen bonds partly or fully broken),with base pair 3 intact (“minor-ld”), illustrated by ApC“BppGCpCpT (Figure 7b). In this structure, the biphenyl is deeply buried in the groove, with close contacts to the bottom (H-1’ and or H-4’) of the 3’-sugar residue of each chain. (3) Base pair 3 distorted (the bases at an angle with hydrogen bonds partly or fully broken), while base pair 1 is intact (“minor-3d”). This is illustrated by TpCPPpT-ApCpA (Figure 7c). The biphenyl has good crossstrand contacts but poor ones on the side facing the modified strand. (4) Distorted, with base-carcinogen cross-strand stack (“minor-bc”),illustrated by CpWPpT*TpCpG(Figure 7d). In this structure, the proximal ring of biphenyl stacks with the cytosine partner of the modified guanine. The hydrogen bonds of base pairs 1and 3 remain intact. In Table 2, the classification of the most stable minor groove adduct is given for each trimer sequence, and its stability with respect to the global minimum. Global minima were divided into three types: major groove, minor groove, or “rearranged”. The last category includes all conformers where one or more bases have moved far from normal double-helical position: see Rearranged Trimers section below. Due to the multiple minimum problem, we cannot be sure that all relevant minima have been identified. Our results thus far, however, suggest that the 5’-neighbor plays a greater role in determining the relative stability of the major and minor groove adducts, with a 5’-purine favoring the major groove structure and a 5’-pyrimidine favoring the minor groove structure (see Table 2). Guanine-Substituted Minor Groove Adducts Nonamer Sequence. We wished to compare minor groove guanine- and adenine-substituted nonamers in the same sequence. The most stable minor groove conformer of GG*T-ATC (an undistorted structure, 3.8 kcaVmol less stable than the major groove global) was embedded in a B-DNA nonamer of the following sequence: GpGpGpGpGABPpTpGpTpG.CpApCpApCpCpCpCpCand energy minimized (see Methods). The angles of the trimer changed little on incorporation into the nonamer. The torsional angles of the central base pairs of the nonamer are included in Table 1, and the nonamer is illustrated in Figure 8. In this conformer, all of the base pairs except the one involving the modified G are intact. The modified

.

124 Chem. Res. Toxicol., Vol. 8, No. 1, 1995

Shapiro et al.

L

Figure 8. Stereoviews of GpGpGpGpGABPpTpGpTpGp.CpApCpApCpCpCpCpC:Minor groove adduct. See the caption to Figure 4. G is roughly coplanar with its partner, but the G and C are not hydrogen-bonded. The biphenyl residue has, on one face, cross-strand contacts with the sugar 3' to the partner of modified G. The other biphenyl face is exposed (no close contacts on the modified strand). This structure is 3.6 kcdmol less stable than the major groove guaninemodified conformer. Adenine-SubstitutedMinor Groove Adducts. Trimer Sequences. For all 16 sequences at the trimer level, a common conformer ("A-minor")with syn adenine could be found which placed biphenyl in the minor groove of a relatively unperturbed B-DNA helix. In these structures (see Table 3 for torsional angles) a' was near 210", and /3' close to 145". The remaining angles of the trimers were within normal B-DNA limits, except for P-2, the pseudorotation of the modified A, which was near 135" (Cl'-exo) and P-5, the pseudorotation of the T opposite the modified A, which was near 40" (C3'-endo). This minor groove structure was the global minimum in 14 of 16 sequences and was much more stable than the major groove structure in all sequences. In Table 4, the stability of the best major and minor groove structures for each sequence are given in kcdmol relative to the global minimum for that sequence. This minor groove conformer is illustrated €or the sequence GpAABppC.ApTpC(Figure 7e). The most prominent defect in the helix is a lack of overlap between bases 5 and 6, which have parallel planes but are not above one another. The ABP rings have cross-strand contacts with sugar 6 and the 2-carbonyl oxygen of cytosine-6, as well as looser contacts to H-1' and H-4' of deoxyribose-3 on the modified strand. Bases 2 and 5 do not display Hoogsteen pairing, but share a single stretched H-bond of 2.25 A from the amino of adenine to 0-2 of thymine (this bond was not present in some sequences, for example, GAABT*AAC). Adedne-SubstitutdMinor Groove Adducb. Nonamer Sequence. The coordinates of the "A-minor" GAABPT.ATCtrimer were embedded in a nonamer of the following sequence: GpGpGpGAmPpTpGpTpGCpApCpApTpCpCpCpC (see Methods). The central AT pair of this sequence was found to be a hot spot for mutagenesis in E . coli virus M13mplO after treatment with a reactive derivative of ABP (3).The torsional angles of the trimer changed only modestly upon incorporation into the nonamer (the torsional angles of the central base pairs of

the nonamer are included in Table 3). In the nonamer, the modified A and its partner share one H-bond of length 2.19 A,from 0-2 of the partner T and the amino of modified A (a type of bonding that we call "wedge"; see Figure 31, rather than a Hoogsteen bond fiom 0-4 of T to amino of A. The ABP residue fits snugly into the minor groove of the nonamer, and the T opposite the modified A has partial overlap with its 3'-neighbor (this was absent at the trimer level). This structure is illustrated in Figure 9. As we mentioned above, this nonamer was 13.2 kcdmol more stable than the major groove A-modified nonamer. Rearranged Trimers. Structures in which there is intercalation of one base into the stack on the other strand, or in which one or more bases move out of position into or beyond the plane of their normal neighbors, have been classified as "rearranged". In several sequences for both the guanine and adenine trimer adducts, the global minimum is a novel structural type within this group,which we call "stack of 4". This subgroup includes the sequences: CpW*pGCpCpG, T p W P ~ T p C p ACpWp, pA*TpCpG, ApGABPpA*TpCpT, CpAABPpA*TpTpG, and TpAABppA*TpTpA.In most cases, the sequence involves a pyrimidine 5' to the modification and a purine 3' to it. The subgroup has modified guanine or adenine anti, a' usually at 200" or higher, rather than near 170", as in the major groove series, and €1 near 200" (in the major groove series, this is near 180"). The backbone angles are generally in the B-DNA range, but the structure is far from a double helix. Its most noteworthy feature is a cross-strand stack involving bases 6,5,2, and 3, which are usually a purine, the pyrimidine partner of the modified G or A, the modified base, and its purine 3' neighbor. This is illustrated for the structure %GABppA*TpCpA(Figure 44. In this structure, the aminobiphenyl ring stacks with the ring and the methyl group of thymine-4 and also with sugar-1. Base pairs 1and 3 are buckled, with hydrogen bonds broken in part. Some structures of this class were unusually stable at the trimer level. For example, TpCPppA*TpCpAwas over 7 kcal/mol more stable than any other conformer of that sequence, and CpGABPpA.TpCpGwas more stable by over 8 kcdmol than the other conformers in its sequence. An attempt was made to build TpGABPpA*TpCpAup to a nonamer. The resulting conformer (data not shown) was over 100 kcdmol less stable than an isomeric major

DNA Trimers Modified by 4-Aminobiphenyl

Chem. Res. Toxicol., Vol. 8, No. 1, 1995 126

Figure 9. Stereoviews of GpGpGpGpAABPpTpGpTpGpApCpApTpCpCpCpC:Minor groove adduct. See the caption to Figure 4. groove adduct structure and was not explored further. An N M R study of a ABP-modified 15-mer containing the above sequence around the modification site found the major groove structure to be the predominant one (5). The "stack of 4" structure may be a curiosity whose stability is limited t o very short DNA double strands. Several other trimer structures (none of them global minima) were also classified as rearranged because they involved cross-strand base-base stacks or one or more bases moved far from normal double-helical position. Some involved syn-modified guanine. They were usually generated in trials in which no penalty function was applied to locate interstrand base pairs (see Methods). These structures were not investigated further.

Discussion Our search of ABP-modified trimers has revealed a striking difference between the guanine and adenine series in the relative stabilities of their major and minor groove adducts. In the guanine adduct series, the two types of conformers were roughly competitive in stability. The identity of the global minimum in a particular case depended upon the sequence, with the stability of the major groove adduct being the most important factor. This could vary by as much as 8 kcdmol among isomers with the same G-C and AT content. The same dependence of stability on sequence was also found in the unmodified trimer duplexes. In studies of longer segmenta of DNA, the strength of nearest-neighbor interactions has been found to vary with ionic strength, so this may hold for ABP-modified DNA as well (21). In one experimental study of the conformation of a 15-mer duplex containing guanine modified by aminobiphenyl, two conformers were found to be present. The predominant one (present to 90% or more) had aminobiphenyl in the major groove. The other was not identified, but intercalated or minor groove locations for aminobiphenyl were suggested. No ionic strength study was reported, but the proportions of the conformers varied with temperature, with lower temperatures (over the range 5-67 "C)increasing the proportion of the major groove structure (5).

In the adenine adduct series, the minor groove structure was at least 10 kcdmol more stable than the major groove one, regardless of sequence (see the Results). A predominant stable minor groove conformation could be observed in all seqyences. Several factors appear to contribute to the differences between the modified guanine and modified adenine series of adducts. In the guanine case, the double-helical structure appears intact in the major groove conformer, with three hydrogen bonds retained from modified guanine to cytosine. The biphenyl residue is exposed in the major groove, with only partial contact on one side of the proximal ring to the 5'-neighbor residue. Several distinct minor groove conformer types were obtained in the guanine series. In general, the conformers with the best contacts between the biphenyl and the sides of the minor groove also suffered the greatest deformation in the planarity and hydrogen bonding of the neighboring base pairs, and vice versa. This suggests that there is a tradeoff between the planarity and strength of H-bonding in the base pairs adjacent to the adduct and the extent of contact of the aminobiphenyl with the walls of the minor groove. The modified guanine did not hydrogen-bond to the cytosine opposite it. The modified adenine series, in comparison to guanine, appears to have a poorer major groove conformer and a single, superior minor groove conformer. The major groove structure has decreased stacking interactions due to propeller twisting by the modified base pair, and only one H-bond from modified adenine to thymine. The minor groove series also shows one H-bond from modified A to T (in some sequences) but has good contacts from biphenyl to both walls of the minor groove. The major and minor groove conformers of the adenine and guanine adducts have been modeled in the same (except for the modified pair) nonamer sequence, which corresponds to the sequence around a hot spot reported for mutagenesis by biphenyl at an A-T pair (3). In this sequence, the principal features observed in the trimers were retained in the nonamers (see Figures 5, 6, 8, and 91, and the difference in stability between major and minor groove conformers remained about the same in both series.

Shapiro et al.

126 Chem. Res. Toxicol., Vol. 8, No. 1, 1995

Mutagenic Implications. In a recent study of aromatic amine mutagenesis using plasmid pBR322 in E. coli, it was observed that ABP produced G to T and G to C transversions primarily, but unlike planar amines such as 2-aminofluorene and 1-aminopyrene, it afforded few frameshifts (4). We suggested earlier that the inability of ABP to induce frameshifts was related to its nonplanarity, which would diminish amine to base stacking interactions (20).A recent high resolution NMR and computational study of DNA modified by a benzo[a]pyrene diol epoxide has illustrated how carcinogen-base stacking can stabilize a bulge in a DNA double helix (23, 24). Structures of this type could possibly produce frameshift deletions in vivo. In the present study, we have defined stable competing major and minor groove conformations for aminobiphenyl bound to guanine and a predominant minor groove conformation for biphenyl bound to adenine. In the major groove conformation, biphenyl does not interfere with the hydrogen bonding of guanine, and it is difficult to see how this adduct might contribute directly t o mutagenesis. The minor groove structures are more suggestive. Normal Watson-Crick pairing has been abolished by the rotation of the modified base t o the syn conformation. Under such circumstances, it is plausible that the specificity of hydrogen bonding of the modified base would be diminished and mispairing would be enhanced. Two combined high resolution NMR and theoretical studies of modified DNA 11-mers described mispairs between syn-aminofluorene-modified guanine and adenine (7) or guanine2 partners. In those structures, the modified pair took on a geometry that we term wedge ( I @ , in which the guanine moves (with respect to Hoogsteen position) toward the minor groove, presumably to optimize amine contacts within the minor groove. By analogy, we may presume that aminobiphenyl-modified guanine could mispair with adenine or guanine in a double helix. If we were to assume that the same stability relationships observed in a double helix also hold at the replication fork, then the presence of syn guanine there could offer an increased opportunity for replication errors, with transversions a plausible result. Such mispairs could explain the frequent aminobiphenylinduced G to T and G to C transversions observed by Melchior and co-workers (4). Similar arguments could be made for the minor groove conformer of the aminobiphenyl-modified adenine. It is also possible to visualize wedge-like A to A pairing involving N-7 of modified A and N6 of partner A in this context, which would provide a structural basis for A*T to T-A transversions (3). The predominance of the minor groove conformation in the case of adenine modification would then provide a possible explanation for enhanced mutagenicity by the adenine adducts of ABP. Additional studies of the structure of DNA helices and replication forks with aminobiphenyl-modifiedA or G and a variety of pairing partners will be needed to substantiate these ideas. Acknowledgment. This work was supported by NIH CA28038 (S.B. and R.S.), NIH RR06458 (S.B. and R.S.), U S . DOE FG0290ER (S.B and R.S.), U.S. DOE Contract Abuaf, P., Hingerty, B. E., Broyde, S., and Grunberger, D. (1995) Solution conformation of the N-(deoxyguanosin-8-yl)aminofluorene adduct opposite inosine and guanine in DNA by NMR and computational characterization. Submitted for publication.

DE-AC05-840R21400with Martin-Marietta Energy Systems (B.E.H.), and US. DOE OHER Field Work Proposal ERKP931 (B.E.H.). Computations were carried out at the Department of Energy’s National Energy Research Supercomputer Center at Livermore, CA, and the National Science Foundation’s San Diego Supercomputer Center. We thank Wilma K. Olson and A. R. Srinivasan for computing the salt concentration employed in DUPLEX.

References Beland, F. A., and Kadlubar, F. F. (1990) Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons in Handbook of Experimental Pharmacology, Vol. 94 lI: Chemical Carcinogenesis and Mutagenesis (Cooper, C. S., and Grover, P. L., Eds.) pp 267-325, Springer-Verlag, Heidelberg. Poirier, M. C., and Beland, F. A. (1992) DNA adduct measurements and tumor incidence during chronic carcinogen exposure in animal models: Implications for DNA adduct-based human cancer risk assessment. Chem. Res. Toxicol. 6 , 749-755. Lasko, D. D., Harvey, S. C., Malaikal, S. B., Kadlubar, F. F., and Essigmann, J. M. (1988) Specificity of mutagenesis by 4-aminobiphenyl. A possible role for N-(deoxyadenosin-8-yI)-4-aminobiphenyl as a premutational lesion. J . Biol. Chem. 263, 1542915435. Melchior, W. B., Jr., Marques, M. M., and Beland, F. A. (1994) Mutations induced by aromatic amine adducts in pBR322. Carcinogenesis 16,889-899. Cho, B. P., Beland, F. A., and Marques, M. M. (1992)NMR studies of a 15-mer DNA sequence from a ras protooncogene, modified at the first base of codon 61 with the carcinogen 4-aminobiphenyl. Biochemistry 31,9587-9602. Broyde, S.,Hingerty, B. E. and Srinivasan,A. R. (1985). Influence of the carcinogen 4-aminobiphenyl on DNA conformation. Carcinogenesis 6,719-725. Norman, D., Abuaf, P., Hingerty, B. E., Live, D., Grunberger, D., Broyde, S., and Patel, D. J. (1989) NMR and computational characterization of the N-(deoxyguanosin-8-y1)aminofluorene adduct [(AF)Glopposite adenosine in DNA: (AF)G[synl.A[antil pair formation and its pH dependence. Biochemistry 28,7462-7476. Cho, B. P., Beland, F. A,, and Marques, M. M. (1994) NMR studies of a 15-mer DNA sequence from a ras protooncogene, modified with the carcinogen 2-aminofluorene: conformational heterogeneity. Biochemistry 33,1373-1384. Eckel, L. M., and Krugh, T. R. (1994) 2-Aminofluorene modified DNA duplex exists in two interchangeable conformations. Nature: Struct. Biol. 1, 89-94. Swaminathan, S., Hatcher, J. F., and Pink, J. C. (1992) [32P1Post labelling analysis of DNA-adducts generated from human uroepithelial cells exposed to N- hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl(N-OAcAABP) and the relationship of adduct formation to induction of mutations in hypoxanthine-guanine phosphoribosyl transferase locus. Abstracts, Special AACR Conference: Chemicals, Mutations and Cancer, Banff, Alberta, Dec 7-12, 1992, p A-33. Bookland, E. A., Swaminathan, S., Oyasu, R., Gilchrist, K. W., Lindstrom, M., and Reznikoff, C. A. (1992) Tumorigenic transformation and neoplastic progression of human uroepithelial cells after exposure in vitro to 4-aminobiphenyl or its metabolites. Cancer Res. 62.1606-1614. (12) Yu, S., Heflich, R. H., Von Tungeln, L. S., El-Bayoumy, IC, Kadlubar, F. F., and Fu, P. P. (1991) Comparative direct-acting mutagenicity of 1-and 2-nitropyrene: Evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts. Mutat. Res. 250, 145-152. (13) Stanton, C. A., Garner, R. C., and Martin, C. N. (1988) The mutagenicity and DNA base sequence changes induced by l-nitroso- and 1-nitropyrene in the CI gene of lambda prophage. Carcinogenesis 9,1153- 1157. (14) Manjanatha, M. G., Newton, R. K., Mittelstaedt, R. A., Villani, G. R. D., Declos, K. B., and Heflich, R. H., (1993) Molecular analysis of DNA adducts and hprt mutations produced by 6-nitrochrysene in Chinese hamster ovary cells. Carcinogenesis 14, 1863-1870. (15) Hingerty, B. E., Figueroa, S., Hayden, T., and Broyde, S. (1989) Prediction of DNA structure from sequence: a build-up technique. Biopolymers 23,1195-1222. (16) Srinivasan, A. R., and Olson, W. (1980) DNA Conformations in real solutions-a preliminary theoretical estimate.Fed. Proc., Fed. Am. SOC.Exp. B i d . 39,2199.

DNA Trimers Modified by 4-Aminobiphenyl Taylor, E., and Olson, W., (1983) Theoretical studies of nucleic acid interactions I. Estimates of conformer mobilities in intercalated chains. Biopolymers 22, 2667-2702. Shapiro, R., Hingerty, B. E., and Broyde, S.(1989) Minor groove binding models for acetylaminofluorenemodified DNA. J.Bioml. Struct. Dyn. 7 , 493-513. Amott, S., Smith, P. J. C., and Chandrasekaran, R. (1976)Atomic coordinates and molecular conformations for DNA-DNA, RNARNA and DNA-RNA helices. In Handbook of Biochemistry and Molecular Biology, 3rd ed., Vol. 11,Nucleic Acids (Fasman, G. D., Ed.) pp 411-422, CRC Press, Cleveland, OH. Shapiro, R., Underwood, G., Zawadzka, H., Broyde, S., and Hingerty, B. E. (1986) Conformation of d(CpG) modified by the carcinogen 4-aminobiphenyl: A combined experimental and theoretical analysis. Biochemistry 26, 2198-2205. (21) Doktycz, M. L., Goldstein, R. F., Paner, T. M., Gallo, F. J., and Benight, A. S.(1992)Studies of DNA dumbbells. I. Melting curves of 17 DNA dumbbells with different duplex stem sequences linked by T4 endloops: Evaluation of the nearest-neighbor stacking interactions in DNA. Biopolymers 32,849-864.

Chem. Res. Toxicol., Vol. 8, No. 1, 1995 127 (22) Fenley, M. O., Manning, G. S.,and Olson, W. K. (1990)Approach to the limit of counterion condensation. Biopolymers SO, 11911203. (23) Cosman, M., Fiala, R., Hingerty, B. E., Amin, S., Geacintov, N. E., Broyde, S.,and Patel, D. J. (1994) Solution conformation of the (+>trans-anti-[BPldG adduct opposite a deletion site in a DNA duplex: Intercalation of the covalently attached benzo[alpyrene into the helix with base displacement of the modified deoxyguanosine into the major groove. Biochemistry 33,11507-11517. (24) Cosman, M., Fiala, R., Hingerty, B. E., Amin, S.,Geacintov, N. E., Broyde, S., and Patel, D. J. (1994) Solution conformation of the (+)-cis-anti-[BPIdG adduct opposite a deletion site in a DNA duplex: Intercalation of the covalently attached benzo[ulpyrene into the helix with base displacement of the modified deoxyguanosine into the minor groove. Biochemistry SS,11518-11527. (25) Altona, A. and Sundaralingam, M. (1972)Conformationalanalysis of the sugar ring in nucleosides and nucleotides.A new description using the concept of pseudorotation. J. Am. Chem. SOC. 94, 8205-8212.

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