A Slipped Replication Intermediate Model Is Stabilized by the Syn

Chem. Res. Toxicol. 1998, 11, 1301-1311

1301

A Slipped Replication Intermediate Model Is Stabilized by the Syn Orientation of N-2-Aminofluorene- and N-2-(Acetyl)aminofluorene-Modified Guanine at a Mutational Hotspot Debjani Roy,† Brian E. Hingerty,‡ Robert Shapiro,*,§ and Suse Broyde*,† Biology Department, New York University, New York, New York 10003-5181, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830-6480, and Chemistry Department, New York University, New York, New York 10003-5180 Received May 18, 1998

The Escherichia coli NarI restriction enzyme recognition site 5′G1G2C3G4C5C63′ is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5′GGCC3′ when G4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 41474151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G4 by the related aromatic amine 2-aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., KoffelSchwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I: 5′ G1 G2 C3 G4 C5 C6 A7 3′ 3′ C11 G10 G9 T8 5′ Intermediate II: 5′ G1 G2 C3 G4 C5 C6 A7 3′ 3′ C11 G10 -------- G9 T8 5′

Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.

Introduction Aromatic amines are found in the environment as byproducts of fossil fuel combustion, in tobacco smoke, and in barbecued meat and fish (1-4). N-2-Aminofluorene (AF)1 and N-2-(acetyl)aminofluorene (AAF) are model aromatic amines whose mutagenic and tumori* Corresponding authors. S.B.: phone, (212) 998-8231; fax, (212) 995-4015; e-mail, [email protected]. R.S.: phone, (212) 998-8484; fax, (212) 260-7905; e-mail, [email protected]. † Biology Department, New York University. ‡ Oak Ridge National Laboratory. § Chemistry Department, New York University. 1 Abbreviations: AF, N-2-aminofluorene; AAF, N-2-(acetyl)aminofluorene.

genic properties have been under investigation for more than half a century (1, 5-8). Following their metabolic activation, AAF and AF bind to the C8 position of guanine, forming N-(deoxyguanosin-8-yl)-2-(acetyl)aminofluorene as the major AAF adduct and N-(deoxyguanosin-8-yl)-2-aminofluorene as the only AF adduct (Figure 1). In vivo, the AAF adduct is largely enzymatically deacetylated to the AF one (1). An extensive review of the AF and AAF mutagenicity literature has been presented by Heflich and Neft (6). High-resolution NMR solution structures of oligonucleotides modified by these adducts have recently been reviewed by Patel et al. (9). DNA replication errors leading to frameshift mutations are most prevalent in contexts such as repetitive se-

10.1021/tx980106w CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998

1302 Chem. Res. Toxicol., Vol. 11, No. 11, 1998

Figure 1. Structures of (A) N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and (B) N-(deoxyguanosin-8-yl)-2-(acetyl)aminofluorene (dG-C8-AAF) and definitions of linkage site torsion angles (A-B-C-D). The angle A-B-C-D is measured by a clockwise rotation of D with respect to A, looking down the B-C bond. A eclipsing D is 0°. (A) For AF, R′ is N9(G)-C8(G)-N(AF)-C2(AF) and β′ is C8(G)-N(AF)-C2(AF)-C1(AF). (B) For AAF, R′ is N9(G)-C8(G)-N(AAF)-C2(AAF), β′ is C8(G)-N(AAF)-C2(AAF)-C1(AAF), γ′ is C8(G)-N(AAF)-C(AAF)Cm(AAF), and δ′ is N(AAF)-C(AAF)-Cm(AAF)-Hm(AAF). Cm and Hm represent the acetyl methyl group. χ (purine) is O4′C1′-N9-C4.

quences that permit template-primer misalignment (10-16). It is noteworthy that carcinogen modification can markedly enhance the propensity of a given slippageprone sequence to produce frameshift mutations (17-20). Polymerase pausing in the vicinity of the damaged base, which permits time for the slipped mutagenic intermediate to form, is believed to play a key role in this mutagenicity enhancement (7, 21, 22). The Escherichia coli NarI restriction enzyme recognition site 5′G1G2C3G4C5C63′ is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5′GGCC3′ when G4 is modified by AAF (7, 17, 18, 23-28), even though each G shows a similar reactivity (29). Site-specific modification studies in pBR322 of E. coli (30) and in human cells (31) confirmed that the -2 deletion definitely results only from modification at the third G of the sequence. The spontaneous level of -2 frameshift mutations at the NarI site is in the range of 10-8 (19). A single AAF adduct increases the -2 mutation frequency up to 10-1 (32). Modification at G4 by the related AF, which lacks the acetyl group of AAF, also enhances -2 deletions, although it is about 10-fold less efficient than AAF in inducing these mutations (18, 24). Computer modeling studies suggested in 1987 that AAF- and AF-induced -2 frameshift mutations could arise from a slippage mechanism in a repetitive GC sequence (33). A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication (14, 32, 34). In this model (Figure 2), the replication apparatus is delayed before the modified guanine residue, but eventually incorporates a cytosine opposite it. Pausing of the replication fork at the modified guanine residue would favor slippage of the nascent strand and leave two bases of the NarI sequence looped out in the parental stand. This misaligned intermediate contains two terminal

Roy et al.

Figure 2. Replication slippage mechanism as proposed by Schaaper et al. (14).

bases in the primer (3′CG5′) hydrogen bonded with a repeated downstream complementary 5′GC3′ dinucleotide in the template. Continued synthesis from this intermediate, which now contains a two-nucleotide bulge in the template strand, leads to a newly synthesized strand two bases shorter than the template strand. The model explains why -2 deletion mutagenesis occurs at at G4 but not at the G1 or G2 position of the NarI site, since only G4 would allow formation of a misaligned intermediate stabilized by two correct terminal base pairs. Belguise-Valladier and Fuchs (35) have studied the single-stranded NarI sequence modified with AAF and AF at G1, G2, or G4 (designated as G1, G2, and G3 in their paper, respectively) with chemical probing experiments and in vitro primer elongation assays. Translesion synthesis is found with all polymerases tested (Pol III holoenzyme and exo+ and exo- Klenow fragments, Sequenase 2.0) when the AF adducts are at G1 or G2, while little bypass is seen when the AF adduct is at G4. On the other hand, AAF adducts block DNA synthesis irrespective of their position within the NarI sequence in the in vitro system (36). Chemical probing experiments suggested that AF adducts at G1 and G2 are in most cases nondistorting adducts and as a consequence do not block replication. The deformation induced by the AAF at G1 and G2 is larger than the distortion induced by the AF lesion. But at the third position of the NarI site, the AF adducts “behave almost as AAF adducts in terms of structural distortion induced and its replication blockage property” (35). The key question that we address in this study is why does carcinogen modification dramatically increase the propensity for mutagenicity at the hotspot? Is there some conformational feature induced by the carcinogen that enhances the likelihood of slippage? Also, why does AAF enhance the mutagenicity more than AF? To address the above questions, we studied the following intermediates which model replication at one arm of a fork in a fragment of DNA modified by AF or AAF (designated by *) at the G4 hotspot in the NarI sequence. ∗

AF- and AAF-Stabilized Mutagenic Structures

Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1303

Intermediate I models the situation in which the replication machinery correctly incorporates cytosine opposite the G4 hotspot, which may lead to the misaligned intermediate, shown in intermediate II. ∗

In this structure, a misaligned slipped mutagenic intermediate has formed where the two terminal bases of the primer (3′C11G105′) hydrogen bond with a repeated downstream complementary sequence (5′G2C33′) in the template. Extension of this intermediate gives rise to the double-base frameshift mutation product. Recently, we reported results for intermediates I and II without carcinogen modification (37). As expected for faithful replication, which is the predominant event in the unmodified NarI sequence (19, 34), the G4 anti normally extended intermediate I is strongly favored over all intermediate II conformations. Strikingly, however, we also found that bulged structures (intermediate II) and their normally extended counterparts (intermediate I) are about equal in energy when both are in the syn conformation of G4. These results suggested that in the presence of a carcinogen which stabilizes the syn conformation, the bulge and the extended form might both be syn and have similar energies.

Methods Force Field and Geometry. We employ the torsion angle space molecular mechanics program DUPLEX to compute energy-minimized structures of the replication intermediates (38). DUPLEX employs a consistent force field for nucleic acids based on one devised in the Olson laboratory (39, 40) which contains the usual van der Waals, electrostatic, and torsional contributions to the energy Enb, Eel, and Etor, respectively, as well as a number of special terms. These are needed to properly model sugar conformations (41) and phosphodiester rotational preferences (42). The original treatment for hydrogen bonding devised by Olson (43) is also employed. Eel is an especially important term in the force field because electrostatics play a central role in determining the molecular structure of the DNA. We employ a distance-dependent dielectric function (r) which models the interpenetration of solvent as the distance r between an atom pair increases ( ) ′/e-βr) (39, 40). The choice for the value of β in the screening term of the dielectric function allows any selected concentration of counterion to be represented (44). β is assigned a value of 0.1 in this work which corresponds to screening by a monovalent salt concentration of about 0.1 M (44, 45). The choice of ′, a variable parameter, governs the exact shape of the function. In this work, we employ a value of 1 on the basis of earlier experience in our laboratory.2 In addition, reduced partial charges on the pendant phosphate oxygens are employed to model charge neutralization by counterion condensation (46). The DNA geometry is taken from Arnott et al. (47). Geometries and force field parameters for the AF and AAF adducts are taken from previous work (48-50). Search Strategy. (1) Hydrogen Bonding Patterns. A feature of DUPLEX that played a major role in this work is the hydrogen bond penalty function which provided an important tool for searching for the different types of structures. This penalty function or pseudopotential energy, F, is added to the energy, and is employed to locate minimum energy conformations of any designated hydrogen bonding pattern, or a denatured site if the function is not employed at a given position. 2

R. Shapiro, S. Ellis, and S. Broyde, unpublished data.

Figure 3. Protonated hydrogen bonding patterns that were investigated: (A) Hoogsteen and (B) Wedge. The penalty function has the form n

F)W

∑[(d - d ) o

2

+ [1 + cos(τ)2 + p]]

i)1

W is an adjustable weight, and values of W are in the range of 5-50 kcal mol-1 Å-2) depending on n, the number of targeted hydrogen bonds. d is the value of the current donor-acceptor distance, and do is the value of the ideal target distance. The current angle around the hydrogen atom, the donor-hydrogenacceptor angle, is τ. p ) |c1 - c2|2, where c1 is a unit vector perpendicular to the plane of one base and c2 is a unit vector perpendicular to the plane of its partner. In an ideal hydrogen bond, d ) do, τ ) 180°, c1 is parallel to c2, and F, which is summed over all n hydrogen bonds at all residues, equals 0. The hydrogen bond penalty function F is employed in the first stage of minimization. A second minimization is then carried out in which the function is released so that final structures are unrestrained energy minima. The function can be applied to any chosen donor-hydrogen-acceptor pair in Watson-Crick, Hoogsteen, or any other type of selected hydrogen bonding scheme. If the function is not applied to a given base pair, a denatured site can result. It should be emphasized that the hydrogen bonding scheme which is targeted by the hydrogen bond penalty function may or may not be achieved during the stages of minimization, and a final unrestrained structure may have a different hydrogen bonding pattern than the one that was initially searched for. (2) Starting Structures for AF-Modified DNA. Minimized unmodified intermediates (37) were employed as starting conformations for the DNA in the corresponding AF-modified trials. The AF was positioned in 16 different starting conformations of the torsion angles R′ and β′ which govern the position of the AF in relation to the guanine residue (Figure 1), dividing the 360° space of R′ and β′ into quadrants (90°), in combination with syn and anti glycosidic torsions: χ ) 240° (anti) or 60° (syn); R′ ) 0°, 90°, 180°, or 270°; and β′ ) 0°, 90°, 180°, or 270° in all 32 combinations. The hydrogen bond penalty function was then employed in the following way. At residues with normal Watson-Crick pairing, we employed the function to seek out that type of bonding. In bulged structures, the function was employed only for the paired bases, and the nonpaired bases were not targeted for any hydrogen bonding. In intermediate I where there is a C (C11) opposite the G4 hotspot, five different hydrogen bonding schemes were sought for the G4‚C11 base pair. Three of them are Watson-Crick (G4 anti), Hoogsteen (G4 syn), and Wedge (G4 syn) (Figure 3). The other two involved syn G4 opposite anti C11 and anti G4 opposite anti C11, with no hydrogen bonding between G4 and C11. (3) Starting Structures for AAF-Modified DNA. Minimized syn AF-modified replication intermediates were used as the starting conformations for the corresponding AAF-modified trials, except in a few selected cases (see the Results). The anti orientation is strongly disfavored in previous computations for


Roy et al.

Table 1. AF-Modified Anti Major Groove Structures of Intermediate Ia minimum no. energy (kcal/mol) 1 2 3

-329.0 -326.7 -322.9

∆E 0.3 2.6 6.4

χ

R′

β′

240 112 50 243 121 226 239 108 176

Table 2. AF-Modified Syn Minor Groove Structures of Intermediate Ia

Figure

minimum no.

energy (kcal/mol)

∆E

χ

R′

β′

Figure

4A ns ns

1 2 3 4

-329.3 -324.6 -323.6 -323.3

0.0 4.7 5.7 6.0

36 59 68 69

220 214 207 179

27 56 315 75

4B 4C ns ns

a Computed structures to 8 kcal/mol are shown. ns means not shown; see the text for description.

AAF modification even in small DNA subunits, due to steric hindrance between the bulky acetyl group and the sugarphosphate backbone linked to the modified guanine (51). The AAF specific torsion angles γ′ and δ′ which determine the position of the acetyl group of AAF in relation to the ring structure (Figure 1) had to be assigned starting values. γ′, the torsion angle between the amine nitrogen and the carbonyl carbon, was assigned values of 0° and 180°. For δ′, the torsion angle between the carbonyl carbon and the methyl carbon, a starting value of 60° was assigned. These choices were made on the basis of earlier work which had delineated the favored domains for these torsions (51). Thus, we created two different starting conformations for AAF-modified DNA (γ′ ) 0° and δ′ ) 60°, and γ′ ) 180° and δ′)60°) from each of the corresponding AF-modified ones. (4) Additional Trials. We have also used interactive computer graphics to create some additional starting structures. Energy minima obtained from the trial sets for AF and AAF modification were in some cases manipulated using the visualization program Insight II from Molecular Simulations Inc. to generate improved models for further energy minimization. Also, for each lowest-energy AF- and AAF-modified structure up to 5 kcal/mol from the lowest-energy conformation, we turned β′ by 180°, corresponding to a 180° flip about the AF and AAF long axis, and reminimized to specifically seek the β′ turned conformer. (5) Total Number of Trials. A total of about 1000 structures for AF-modified intermediates and about 85 structures for AAF-modified intermediates were generated and evaluated. (6) Energy Minimization. The energy minimizations were carried out in stages. The hydrogen bond penalty function with a weight of 15 was employed in all first-stage minimizations to guide in the location of the selected hydrogen bonding patterns. A second minimization was performed for each resulting structure from the first minimization without the hydrogen bond penalty function. Those structures that employed protonated bases (Hoogsteen and Wedge hydrogen bonded structures, Figure 3) were deprotonated and minimized again to permit comparison of their energies with those of the unprotonated conformers (52). The energy-minimized structures resulting from each intermediate were ranked according to energy. Computations were carried out on a Cray C90 Supercomputer at the National Science Foundation San Diego Supercomputer Center and on a Cray C90 computer at the Department of Energy National Energy Research Supercomputer Center. The computer graphics program Insight II from Molecular Simulations Inc. was used for visualization on SGI workstations.

Results The energies of intermediates I and II are directly comparable since they are in fact different conformers of the same molecule. Energies (∆E) of a structure are given relative to the lowest-energy form, regardless of which intermediate provided the structure with the lowest energy. The results given below and elaborated in the Discussion reveal that intermediates I and II can have structures with comparable low energies. In the AF case, structures in which the modified guanine is syn or anti are competitive in intermediate I, but a syn structure is favored for intermediate II. In the AAF case,

a Computed structures to 8 kcal/mol are shown. ns means not shown; see the text for description.

syn structures are the most stable for both intermediates I and II, and anti is strongly disfavored.

∗

AF-Modified Anti Major Groove Structures of Intermediate I. Structures with AF in the major groove and modified guanine anti provided an important family of structures for this intermediate. In the most favored major groove structure (Table 1, no. 1, ∆E ) 0.3 kcal/ mol, Figure 4A), the G4 hotspot is anti and the G4‚C11 Watson-Crick base pair is nicely stacked with the C5‚ G10 base pair above it. The AF points in the 3′ direction along the modified strand, toward the duplex region. The AF is partly stacked with the single-stranded region on one face and mainly exposed on the other, with the C9containing edge of the AF directed inward. The sugarphosphate backbone between G4 and C3 makes a sharp turn, and all three bases in the single-stranded region are stacked with each other. A variant of Figure 4A with β′ rotated ∼180° has the C9 edge directed outward (Table 1, no. 2, ∆E ) 2.6 kcal/mol). Another variant, whose energy is 6.4 kcal/mol has the AF mainly exposed on both faces and the C9-containing edge directed outward. AF-Modified Syn Minor Groove Structure of Intermediate I. Structures with AF in the minor groove and modified guanine syn provided another important family of structures for this intermediate. In the most favored minor grove structure (Table 2, no. 1, ∆E ) 0.0 kcal/mol, Figure 4B), the G4 hotspot is syn and the AF is oriented 3′ along the modified strand toward the duplex region. One face of the AF is almost completely exposed. The other face of the AF is shielded by the sugarphosphate backbone of the modified strand. The C9 edge of the AF is directed toward the base-paired region. No hydrogen bond is formed between G4 and C11. C3 is stacked with G2, and G1 is partly stacked with G2. The sugar-phosphate backbone between G4 and C3 makes a U-turn. The second conformer of this type (Table 2, no. 2, Figure 4C) differs mainly in that the single-stranded region curves toward the primer strand. The third conformer (Table 2, no. 3) resembles the second conformer except that the C9 edge of the AF is directed outward. The fourth conformer (Table 2, no. 4) is a variant of the second with AF positioned somewhat closer to the unmodified strand. AF-Modified Syn Inserted Structure of Intermediate I. The third structural family found for this intermediate is base displaced-intercalated with modified guanine syn. In the lowest-energy variant of this type (Table 3, no. 1, ∆E ) 6.7 kcal/mol, Figure 4D), the G4 is syn, displaced in the major groove, and the G4‚C11 base



Figure 4. (A) AF-modified anti major groove conformation of intermediate I. ∆E ) 0.3 kcal/mol (Table 1, no. 1). (B) AF-modified syn minor groove conformation of intermediate I. ∆E ) 0.0 kcal/mol (Table 2, no. 1). (C) AF-modified syn minor groove conformation of intermediate I. ∆E ) 4.7 kcal/mol (Table 2, no. 2). (D) AF-modified syn inserted conformation of intermediate I. ∆E ) 6.7 kcal/mol (Table 3). The stereoviews are color-coded as follows: modified template strand, magenta; primer strand, dark blue; AF, green; and G4 hotspot, yellow. Table 4. AF-Modified Structures of Intermediate IIa

Table 3. AF-Modified Syn Inserted Structure of Intermediate Ia minimum no.

energy (kcal/mol)

∆E

χ

R′

β′

Figure

1

-322.6

6.7

54

261

90

4D

a

Computed structures to 8 kcal/mol are shown.

pair is disrupted. The AF is inserted between G10 and C11, and stacked on one face with the C5‚G10 pair. On the other face, the AF is partly in contact with C11 and partly exposed. The C9 edge of the AF is directed toward the single-stranded region. The bases in the singlestranded region are stacked.

∗

All low-energy structures for this intermediate contained syn modified guanine. The lowest-energy anti structures we found had a ∆E of 31.2 kcal/mol (see the Discussion). In the lowest-energy structure of this type (Table 4, no. 1, ∆E ) 1.1 kcal/mol, Figure 5A), the G4 is syn and protrudes into the major groove. On one face, the AF is partly stacked with the C3‚G10 pair, and on the other face, it is partly stacked with the sugar-phosphate backbone and partly exposed. The C9 edge of the AF is directed toward the unmodified strand. The C5 without a partner is stacked with the C6‚G9 base pair. The C6‚

minimum no.

energy (kcal/mol)

∆E

χ

R′

β′

Figure

1 2

-328.2 -322.3

1.1 7.0

71 54

151 256

144 264

5A 5B

a


G9 and A7‚T8 base pairs are distorted. The helix axis between the two base-paired regions is shifted, resulting in a bend which creates a pocket for the protruding G4. In the second lowest-energy structure of this type (Table 4, no. 2, ∆E ) 7.0 kcal/mol, Figure 5B), the G4 is again syn and in the major groove. The AF on one face is stacked with the C6‚G9 pair, and on the other face, it is mainly exposed in the minor groove. The C9 edge of the AF is directed toward the unmodified strand. The C5 without a partner is in the minor groove with one face exposed. The C6‚G9 and A7‚T8 base pairs are normal. The helix axis between the two base-paired regions is shifted somewhat, resulting in a modest bend which again provides a binding pocket for the AF and C5.

∗

AAF-Modified Anti Structure of Intermediate I. Our search strategy was mainly limited to seeking out syn conformers for the AAF-modified intermediates (see


Roy et al.

Figure 5. AF-modified syn conformation of intermediate II. (A) ∆E ) 1.1 kcal/mol (Table 4, no. 1). (B) ∆E ) 7.0 kcal/mol (Table 4, no. 2). The stereoviews are color-coded as follows: modified template strand, magenta; primer strand, dark blue; AF, green; G4 hotspot, yellow; and C5 without a partner, cyan. Table 5. AAF-Modified Syn Minor Groove Structure of Intermediate Ia minimum no. energy (kcal/mol) ∆E χ 1 a

-341.5

R′

β′

γ′

Table 6. AAF-Modified Syn Inserted Structure of Intermediate Ia

δ′ Figure

5.0 69 234 50 200 27

6A


the Methods), since the anti conformation is strongly disfavored due to steric hindrance in this adduct (51). However, for completeness, we did use the lowest-energy anti conformers of AF intermediates I and II as AAF adducts (with γ′ ) 0° and 180° and δ′ ) 60°) as starting conformations for energy minimizations. In intermediate I, the major groove anti structure for the AAF adduct had a very high energy (∆E ) 42.8 kcal/mol), as expected. AAF-Modified Syn Minor Groove Structures of Intermediate I. In the AAF case, intermediate I has a somewhat higher energy than intermediate II. The lowest-energy structure of intermediate I (Table 5, no. 1, ∆E ) 5.0 kcal/mol, Figure 6A) resembles somewhat the minor groove AF structure shown in Figure 4C. AAF is oriented 3′ along the modified strand, toward the duplex region in the minor groove. One face of the AAF is shielded by contact with the base edges of the doublestranded region at G4‚C11 and C5‚G10. The other face of the AAF is exposed. The C9 edge of the AAF is directed toward the modified strand, and the methyl group of the AAF is directed outward. The carbonyl oxygen of the AAF is oriented toward the sugar-phosphate backbone of C3. The modified guanine is syn and stacked with C5. At the G4‚C11 base pair, one hydrogen bond is formed (O6 of G4 with the amino hydrogen of C11). The three unpaired bases in the single-stranded region are stacked. There is a bend between the duplex and single-stranded region at G4 and C3.

energy (kcal/mol) ∆E 1 AAF a

-338.8

χ

R′

β′

γ′

δ′

Figure

7.7 57 268 87

9

33

6B


AAF-Modified Syn Inserted Structure of Intermediate I. The inserted AAF intermediate of Table 6 (no. 1, ∆E ) 7.7 kcal/mol, Figure 6B) is similar to the related AF structure shown in Figure 4D. In the AAF case, the acetyl methyl group is in contact with one face of C3 and the carbonyl oxygen is directed outward.

∗

The lowest-energy structure of this intermediate (Table 7, no. 1, ∆E ) 0.0 kcal/mol, Figure 7) bears resemblance to the related AF structure shown in Figure 5B. The G4 is syn and protrudes into the major groove. The AAF on one face is partly stacked with the C6‚G9 pair and also is in contact with the sugar-phosphate backbone between residues C6 and A7. On the other face, it is partly stacked with the sugar-phosphate backbone of C11 and G10 and partly exposed. The C9 edge is directed toward the modified strand. The acetyl methyl group of AAF is directed toward the modified strand, and the carbonyl oxygen of the AAF is directed outward. The unpartnered C5 is completely exposed on one face, and on the other face, it is partly stacked with the C9-containing edge of the AAF. The helix axis between the two base-paired



Figure 6. (A) AAF-modified syn minor groove conformation of intermediate I. ∆E ) 5.0 kcal/mol (Table 5, no. 1). (B) AAF-modified syn inserted conformation of intermediate I. ∆E ) 7.7 kcal/mol (Table 6). The structures are color-coded as follows: modified template strand, magenta; primer strand, dark blue; AAF, green; and G4 hotspot, yellow.

Figure 7. AAF-modified syn conformation of intermediate II. ∆E ) 0.0 kcal/mol (Table 7, no. 1). The stereoview is color-coded as follows: modified template strand, magenta; primer strand, dark blue; AAF, green; G4 hotspot, yellow; and C5 without a partner, cyan. Table 7. AAF-Modified Syn Structures of Intermediate IIa minimum no.

energy (kcal/mol)

∆E

χ

R′

β′

γ′

δ′

Figure

1 2 3

-346.5 -346.2 -340.7

0.0 0.2 5.7

56 54 54

244 250 246

112 277 277

13 7 183

30 39 73

7 ns ns

a

Computed structures to 8 kcal/mol are shown. ns means not shown; see the text for description.

regions is shifted somewhat, resulting in a modest bend that provides a binding pocket for the protruding rings of the AAF and C5. Structure 2 of Table 7 (∆E ) 0.2 kcal/mol) is the β′turned version of structure 1 of Table 7 (Figure 7). The C9 edge of AAF is consequently directed toward the

unmodified strand. Structure 3 of Table 7 (∆E ) 5.7 kcal/ mol) is the β′-turned, γ′-turned version of structure 1 of Table 7 (Figure 7). Thus, the methyl group is directed outward and the carbonyl oxygen is directed inward toward the modified strand.

Discussion Comparison with Experimental Results. (1) NMR Solution Studies. Experimental NMR solution studies of AF and AAF adducts reveal observed structures with AF both syn and anti while the AAF adduct is syn (reviewed in ref 9). A much larger body of structures is available for AF than for AAF modification. The AF observed structures are of three types, with a strong sequence dependence governing structural type: AF in the major groove with the modified guanine anti in a


Roy et al.

Table 8. Energies of Intermediate I (Extended) versus Energies of Intermediate II (Bulged) in Lowest-Energy Anti and Syn Conformations energya (kcal/mol)

AF-modified AAF-modified

intermediate I anti

intermediate II anti

intermediate I syn

intermediate II syn

-329.0 (0.3) -303.7 (42.8)

-298.1 (31.2) -304.7 (41.8)

-329.3 (0.0) -341.5 (5.0)

-328.2 (1.1) -346.5 (0.0)

a Absolute energies are followed by ∆E, the energy of the given structure relative to the lowest-energy form, in parentheses. Note that energies of intermediates I and II are directly comparable since they are in fact simply different conformers of the same molecule.

normal duplex (53-57); AF in the minor groove with the modified guanine syn in duplexes with a mismatched A (58), G, or I opposite the AF (59); and AF in a base displaced-intercalated conformation with the modified guanine syn and displaced into the major groove in structures where the guanine glycosidic rotation can be unambiguously determined in a normal duplex (56, 57), in bulged duplexes (60-62), and at single strand-double strand junctions (63, 64). One structure for AF-modified intermediate I (9, 64) is of the syn guanine base displaced intercalated type, but no intermediate II structures are yet available. NMR solution structural studies of AF-modified duplexes in the NarI sequence reveal a strong sequence dependence on conformation, with the proportion of anti major groove structures versus syn base displacedintercalated ones being 70:30, 90:10, and 50:50 when modification is at G1, G2, and G4, respectively (designated as G4, G5, and G7, respectively, in refs 9, 56, and 57). Thus, the proportion of syn AF structures is highest at the G4 hotspot in the duplex. Differing base sequences can also explain the fact that our computed structures for intermediate I contain syn and anti structures with nearly equal energies (Tables 1 and 2), while the NMR solution structure for this intermediate is syn (9, 64). This is especially plausible because the syn-anti equilibrium is so exquisitely sensitive to base sequence that even the next nearest neighboring base significantly influences the population balance (9, 56, 57). A high-resolution AAF structure in a normal DNA duplex is of the syn guanine base displaced-intercalated type (65). NMR data for an AAF-modified bulged duplex in which the modified G is without a partner revealed conformational heterogeneity (66). The data were interpreted to suggest an equilibrium between a conformer in which the AAF is inserted into the helix in ∼30% of the population and external in the remaining ∼70%. NMR studies have also been carried for an AAF-modified NarI sequence duplex containing a two-base bulge in the modified strand (67). Conformational heterogeneity was again manifested. The data were interpreted to be consistent with intercalation of the AAF moiety with displacement of the modified guanine. The AAF-modified guanine was deemed to be syn in both bulged structures (66). (2) Primer Extension Replication Assays and Chemical Modification Studies. Belguise-Valladier and Fuchs (35) found that AF adducts at NarI G4 (designated as G3 in their paper) “behave almost as AAF adducts”. They cause polymerase blockage and distortion when the modification is at G4, but not at G1 or G2 (designated as G3, G1, and G2, respectively, in their paper) where rapid bypass and little distortion is observed. In the AAF case, blockage and distortion are found at all three positions (36). These findings are in line with our results which indicate that the AF adduct

at the G4 hotspot can feasibly adopt AAF-like syn structural types in intermediates I and II. (3) Polymerase Crystal Structures Containing Primer-Template Complexes. An interesting feature of all the important structures of intermediate I modeled in this work is the fact that the DNA is bent and the templating C3 base is not stacked with the previously replicated base. This is in line with a model based on the crystal structure of human DNA polymerase β with a DNA primer-template complex (68) which contains a 90° bend in the single-stranded template. In addition, recent crystal structures of primer-template complexes with T7 DNA polymerase (69) and Bacillus stearothermophilus DNA polymerase I (70) also reveal the existence of such bends which displace the templating base. Doublie et al. (69) state “the template strand enters the polymerase active site from the side away from the thumb (the polymerase domain has a shape reminiscent of a right hand in which the palm, fingers and thumb form a DNA binding groove that leads to the polymerase active site) and a sharp turn exposes the template base for interaction with the incoming nucleotide”. (4) Bulged Structures and Bending. Our intermediate II structures are bent, and bending is known to be associated with bulged unmodified duplexes (71-83). NMR solution structures of carcinogen-modified duplexes containing bulged bases on the modified strand also contain bends (60-62, 84-87). In our computed structures, the bends provide binding pockets for protruding aromatic rings of AF, AAF, or bases without partners. The biological significance of bending in bulged structures is not known. Stabilization of the Slipped Mutagenic Intermediate by Syn Guanine. In the AF-modified normally extended intermediate (intermediate I), we found that the energies of the G4 anti and G4 syn conformations are very close, and AF’s intermediate II structures with syn orientation are also energetically comparable to them (Tables 1-7; summarized in Table 8). In the AAF case, anti structures are disfavored (51) (Table 8), but syn intermediates I and II are again energetically close, with the bulged intermediate II actually being preferred. These results suggest that stabilization of the slipped mutagenic intermediate by the carcinogens occurs because they can induce the syn conformation. Increased stabilization of the syn conformation when C8 of purine is modified by bulky substances has long been known (88). It stems from crowding between the modified base and the sugar ring in the anti conformation. This crowding is not so severe as to preclude the anti conformation in the AF case, but for the bulkier AAF steric hindrance involving the additional acetyl group with the adjacent sugar disfavors the anti region more strongly. It is reasonable that guanine in a syn conformation could more readily slip because of its inability to form normal Watson-Crick base pairs in B-DNA. The syn


slipped intermediates are stabilized by many favorable interactions between the carcinogen and the DNA. When we compared the syn slipped intermediate structures with the anti ones (which are much higher in energy; ∆E > 30 kcal/mol as given in Table 8 and therefore not illustrated), we found that anti slipped intermediates place the carcinogen in the major groove with both faces completely exposed and virtually no carcinogen interaction with DNA. In the syn structures, on the other hand, the carcinogens do interact significantly with DNA bases and backbone (Figures 5A,B and 7). Melting temperature studies of AF- and AAF-modified bulged duplexes (with no single-strand overhang) have revealed stabilization of the bulged duplex by the presence of the adduct, which is unambiguously in the syn conformation for the AF adduct (60-62) and likely to be so for AAF as well (66, 67). Stabilization of the slipped mutagenic intermediate by the adduct increases the likelihood that elongation will occur from a slipped structure rather than from a properly aligned one. Elongation from the slipped mutagenic intermediate with the GpC bulge remaining unreplicated would lead to a -2 frameshift mutation. The greater propensity for AAF to adopt the syn conformation than AF in intermediate I is a plausible explanation for its greater propensity for inducing -2 deletions in the NarI sequence. In the AF case, a lowenergy anti structure as well a syn one for intermediate I is computed; however, in the AAF case, the anti domain is strongly disfavored as shown in Table 8 and computed previously by Shapiro et al. (51). Polymerase pausing to permit time for slippage at the lesion site could be enhanced by an abnormal syn conformation versus an anti one which merely places the carcinogen in the major groove without disrupting Watson-Crick base pairing. Less pausing might therefore be induced by AF and consequently less slippage.

Conclusion In a replicating cellular system, the fork contains many proteins, including polymerase, helicase, single-strand binding proteins, and others. However, an inherent tendency of AF and AAF to increase the stability of the abnormal syn conformation of a modified guanine, compared to an unmodified guanine in the same environment, would remain. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF, which could explain its greater propensity to cause -2 deletions in the NarI sequence.

Acknowledgment. This research is supported by NIH Grants CA 75449, CA 28038, and RR 06458 and DOE Grant DE-FG02-90ER60931 to S.B. and R.S. and DOE Contract DE-AC05-96OR22464 with Lockheed Martin Energy Research to B.E.H. We thank Dr. Robert P. P. Fuchs for very helpful advice and Professor Nicholas E. Geacintov for ongoing interesting discussions.

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TX980106W

A Slipped Replication Intermediate Model Is Stabilized by the Syn

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