Evading the proofreading machinery of a replicative DNA polymerase: induction of a mutation by an environmental carcinogen1

doi:10.1006/jmbi.2001.4674 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 309, 519±536

Evading the Proofreading Machinery of a Replicative DNA Polymerase: Induction of a Mutation by an Environmental Carcinogen Rebecca A. Perlow and Suse Broyde* Department of Biology, New York University, 100 Washington Square East Room 1009M, New York NY 10003, USA

DNA replication ®delity is dictated by DNA polymerase enzymes and associated proteins. When the template DNA is damaged by a carcinogen, the ®delity of DNA replication is sometimes compromized, allowing mispaired bases to persist and be incorporated into the DNA, resulting in a mutation. A key question in chemical carcinogenesis by metabolically activated polycyclic aromatic hydrocarbons (PAHs) is the nature of the interactions between the carcinogen-damaged DNA and the replicating polymerase protein that permits the mutagenic misincorporation to occur. PAHs are environmental carcinogens that, upon metabolic activation, can react with DNA to form bulky covalently linked combination molecules known as carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a common PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and ®sh, and as a contaminant in other foods. BP is metabolically activated into several highly reactive intermediates, including the highly tumorigenic (‡)-antibenzo[a]pyrene diol epoxide (BPDE). The primary product of the reaction of (‡)-anti-BPDE with DNA, the (‡)-trans-anti-benzo[a]pyrene diol epoxide-N2-dG ((‡)-ta-[BP]G) adduct, is the most mutagenic BP adduct in mammalian systems and primarily causes G-to-T transversion mutations, resulting from the mismatch of adenine with BP-damaged guanine during replication. In order to elucidate the structural characteristics and interactions between the DNA polymerase and carcinogen-damaged DNA that allow a misincorporation opposite a DNA lesion, we have modeled a (‡)-ta-[BP]G adduct at a primer-template junction within the replicative phage T7 DNA polymerase containing an incoming dATP, the nucleotide most commonly mismatched with the (‡)-ta-[BP]G adduct during replication. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been carried out, and the resultant trajectory analyzed. The modeling and simulation have revealed that a (‡)-ta-[BP]G:A mismatch can be accommodated stably in the active site so that the ®delity mechanisms of the polymerase are evaded and the polymerase accepts the incoming mutagenic base. In this structure, the modi®ed guanine base is in the syn conformation, with the BP moiety positioned in the major groove, without interfering with the normal protein-DNA interactions required for faithful polymerase function. This structure is stabilized by a hydrogen bond between the modi®ed guanine base and dATP partner, hydrophobic interactions between the BP moiety and the polymerase, a hydrogen bond between the modi®ed guanine base and the polymerase, and several hydrogen bonds between the BP moiety and polymerase side-chains. Moreover, the G:A mismatch in this system clo-

Abbreviations used: T7DNAP, T7 DNA polymerase; BP, benzo[a]pyrene; BPDE, 7,8-dihydroxy-9,10epoxybenzo[a]pyrene diol epoxide; (‡)-anti-BPDE; (‡)-7(R), 8(S),9(S),10(R) BPDE; (ÿ)-anti-BPDE, (ÿ)7(S),8(R),9(R),10(S) BPDE; (‡)-ta-[BP]G, (‡)-trans-anti-[BP]-N2 deoxyguanosine ; 5meC, 5-methyl cytosine; 5meCS, single-stranded 5-methyl cytosine; 5meCT, 50 -terminal 5-methyl cytosine; 5meCD, double-stranded 5-methyl cytosine; SD, steepest descent minimization algorithm; CG, conjugate gradient minimization algorithm; MD, molecular dynamics; PAH, polycyclic aromatic hydrocarbon; ps, picoseconds; ns, nanosecond. E-mail address of the corresponding author: [email protected] 0022-2836/01/020519±18 $35.00/0

# 2001 Academic Press

520

Carcinogen-induced Mismatch in a DNA Polymerase

sely resembles the size and shape of a normal Watson-Crick pair. These features reveal how the polymerase proofreading machinery may be evaded in the presence of a mutagenic carcinogen-damaged DNA, so that a mismatch can be accommodated readily, allowing bypass of the adduct by the replicative T7 DNA polymerase. # 2001 Academic Press

*Corresponding author

Keywords: benzo[a]pyrene; DNA polymerase; mutagenesis; environmental carcinogen; molecular dynamics simulations

Introduction Replicative DNA polymerase proteins invoke multiple proofreading mechanisms to ensure the faithful replication of the genome.1 ± 3 These mechanisms are similar in DNA polymerases from different organisms, including bacteriophage T7,1,4 Escherichia coli (Klenow fragment),5,6 Thermus aquaticus (Taq),7 ± 9 and Bacillus stearothermophilus (Bst).10,11 Despite the proofreading machinery employed by replicative DNA polymerases, carcinogen-damaged DNA is misreplicated more frequently by these enzymes, resulting in an increased mutation rate.12 ± 21 T7 DNA polymerase (T7DNAP), a 698 amino acid residue pol I family polynucleotide polymerase, is an attractive model with which to study DNA replication and replication ®delity because it has been well characterized and requires relatively few cofactors for processive DNA replication. Although other proteins are involved in DNA replication in vivo, the polymerase protein is the catalytic enzyme for the DNA synthesis reaction and requires the recruitment of its E. coli host's thioredoxin protein in order to become a processive enzyme, capable of polymerizing thousands of nucleotides before dissociating from the DNA template.22 To aid in the understanding of DNA replication, speci®cally primer elongation, the three-dimensional structure of the T7DNAP complexed to thioredoxin, a primer-template junction, and an incoming doexynucleoside Ê resolution triphosphate was determined at 2.2 A by Doublie et al.4 Kinetic studies of E. coli DNA polymerase I5,6,23 and T4 DNA polymerase24,25 and then structural studies of several DNA polymerases, including the T7,4 Taq,7 ± 9,26,27 and Bst,11 and mammalian DNA polymerase b28 ± 30 bound to DNA primertemplates, have shown that DNA polymerases undergo a conformational change upon the binding of the correctly matched incoming nucleotide. The structures of these polymerases with and without a bound incoming nucleotide opposite the template base show that the polymerase exists in both an open and a closed conformation. The open conformation is adopted when the polymerase contains the DNA primer-template, but has not yet bound the correctly matched incoming triphosphate, while the closed conformation contains the primer-template and the incoming nucleotide in a conformation that is positioned for the formation

of the new phosphodiester bond. There is a signi®cant motion involved in the conversion of the open to the closed conformation.3,4,29,30 Single-turnover kinetic studies suggest that the rate-limiting step in DNA polymerization occurs after the incoming nucleotide binds to the active site and before the deoxynucleoside triphosphate incomer is incorporated into the growing primer,5,23,31,32 indicating a conformational change as a rate-determining step in primer-elongation. The closing motion has been postulated to represent this change,4 but recent evidence for DNA polymerase b33 suggests that the actual closing motion may be fast, and the rate-limiting step may be the rearrangement of one or more key amino acid residues, at or near the active site needed for catalysis. The transition from the open to the closed conformation of T7DNAP brings critical residues into position for the nucleophilic attack on the incoming nucleotide.4 In addition, the closed conformation of the polymerase active site discriminates against mismatched incoming bases using ``geometric selection''.34 ± 41 An induced-®t mechanism has been proposed in which the polymerase recognizes base-pairs that adopt the correct shape.2,29,30,32,39,41 The movement toward the closed conformation is disfavored in the presence of nucleotide pairs that do not ®t within the parameters required by the discriminating polymerase. Pausing of the polymerization cycle allows the exit of the incorrect deoxynucleoside triphosphate and the continued sampling of the nucleotide pool within the cell until the correct partner is obtained and the polymerase moves into the closed conformation.32,42 In vitro DNA replication studies have shown that the shape of the nascent base-pair is very important in selection of the correct incoming nucleotide, in addition to the actual ®delity of the WatsonCrick hydrogen bonding scheme.2,36 ± 38,43,44 It has been found that interaction between the polymerase and the minor groove of the primer-template DNA complex is required for continued primer extension.43,45 ± 48 The minor groove of the four 30 base-pairs in the primer-template duplex is scanned by contacting residues of the polymerase in order to detect mismatched base-pairs that are sheared with respect to each other or exhibit some other shape that is not characteristic of a correctly matched Watson-Crick base-pair.43 If a mispair is detected, the primer terminus is shifted to the 30 -50 exonuclease site, where the misincorporated base is

Carcinogen-induced Mismatch in a DNA Polymerase

cleaved in order to prevent the propagation of a mutation.1,32,42,43 Mismatches occasionally evade the proofreading machinery, resulting in mutations, which can initiate or contribute to the carcinogenic process if they occur in a gene encoding a protein crucial to cell-cycle control in a multicellular organism, such as an oncogene or tumor suppressor gene.49 ± 52 Polycyclic aromatic hydrocarbons (PAHs) are common environmental chemical carcinogens53 ± 56 that, upon metabolic activation, can react with DNA to form bulky covalently linked combination molecules known as carcinogen-DNA adducts17,53,57 ± 59 The presence of bulky DNA adducts increases the frequency at which incorrect nucleotides are incorporated and mutations occur.13,60 ± 67 When replicating past a bulky adduct, the DNA polymerases often pause or terminate replication due to blockage of the enzyme and the rate and percentage of bypass are dependent on the sequence context of the adduct.61,68 ± 70 Benzo[a]pyrene (BP) is a common environmental PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and ®sh, and as a contaminant in other foods.53 ± 56 BP is metabolically activated71 to form several highly reactive intermediates, including the non-tumorigenic (ÿ)-anti-7,8-dihydroxy9,10-epoxybenzo[a]pyrene diol expoxide (BPDE) and highly tumorigenic (‡)-anti-BPDE.72,73 The primary product of the reaction of the tumorigenic (‡)-anti-BPDE with DNA is the (‡)-trans-anti-N2[BP] deoxyguanosine adduct ((‡)-ta-[BP]G) (Figure 1(a)), which is also the most mutagenic BP adduct in mammalian systems.17,64,67 The (‡)-ta-[BP]G adduct primarily causes G-to-T transversion mutations in a number of replication systems,16 ± 18,21,64,67 including T7DNAP61 (R.A.P., O. Rechokblit, N.E. Geacintov & D.A. Scicchitano, unpublished results), indicating that the bulky

521 modi®ed guanine is mismatched with adenine during replication. There are a number of highresolution NMR solution structures of the (‡)-ta[BP]G adduct in DNA,17,74 ± 78 as well as crystal structures of benzo[a]pyrene itself and certain derivatives.79 ± 81 However, there are no crystal structures of the DNA adduct with or without a DNA polymerase at present. BPDE, together with other metabolically activated environmental PAHs,82 reacts preferentially with mutational hotspots in the human p53 tumor suppressor gene, including hotspot 157, which is speci®c to lung cancer,83 and G-to-T transversion mutations are commonly observed at these hotspots.84 The correlation between the p53 mutational spectrum of human lung tumors and the reactivity spectrum of BPDE in the p53 gene, as well as the fact that the mutations observed within the p53 gene of lung tumors are of the type most commonly caused by BPDE damage, has prompted the association of BP exposure with human lung cancer. Due to this association, it is of much interest to determine the mechanism by which adenine is misincorporated opposite the highly mutagenic (‡)-ta-[BP]G adduct within a processive DNA polymerase. We modeled the (‡)-anti-BPDEdamaged p53 hotspot 157 sequence, as shown in Figure 1(b), because this codon is damaged preferentially by BPDE,83 repaired slowly,84 and is frequently found mutated (G-to-T transversions predominating) in human lung tumors.85 The structure of T7DNAP solved by Doublie et al.4 is an excellent model for studying the replication of a (‡)-ta-[BP]G adduct, because it is one of the few highly processive replicative enzymes for which there is a high-resolution crystal structure containing the polymerase, primer-template model replication fork including the template three base 50 overhang, and the incoming deoxynucleoside triphosphate. In addition, there are experimental data

Figure 1. (a) The structure of the (‡)-ta-[BP]G adduct, including de®nitions of the torsion angles w, a0 , and b0 . w ˆ O40 -C10 -N9-C8; a0 ˆ N1-C2-N2-[BP]C10; b0 ˆ C2-N2-[BP]C10-[BP]C9. (b) The p53 hotspot 157 DNA sequence modeled into the T7 DNA polymerase to create the simulation starting structure. 5meC, 5-methyl cytosine; all CpG sequences within the coding region of the human p53 gene are methylated on the 5 position of the cytosine nucleotide. Note that residue numbers 13 and 14 have not been utilized in the numbering scheme.

522 concerning mutations induced by BPDE adducts during replication by T7DNAP14,61 (R.A.P., O. Rechkoblit, N.E. Geacintov & D.A. Scicchitano, unpublished results) and other polymerases with which it is highly homologous.14 ± 17 The crystal structure gives a clear picture of the conformation of T7DNAP, after it has closed around the incoming nucleotide and is poised to catalyze the addition of the next nucleotide to the primer.4 This structure is in harmony with the proposed induced-®t mechanism by which T7 and other replicative DNA polymerases ensure replication ®delity.30,32,39,41 In order to study the mutagenic replication of (‡)-ta-[BP]G, we modeled the BP-damaged p53 hotspot 157 sequence into the closed conformation of T7DNAP, included a mismatched dATP as the incoming nucleotide, and carried out a molecular dynamics (MD) simulation of this system. This scenario mimics the point at which the enzyme is poised to misincorporate adenine opposite the BPmodi®ed guanine base, resulting in the mutagenic G:A mismatch. Simulating the replication of the p53 hotspot by the T7DNAP, including the mismatched incoming dATP, allowed us to investigate how this particular mismatch evades the extensive scrutiny of the polymerase proofreading machinery. Elucidation of a mechanism permitting a G:A mismatch at the BP-damaged p53 hotspot 157 offers insight into an initiating event that may contribute to carcinogenesis via a mutation in the p53 tumor suppressor gene, and provides insight into the more general mechanism of mutagenic bypass of bulky lesions by replicative DNA polymerases. We chose to use the AMBER 5.0 molecular dynamics simulation package,86 the Cornell et al. force-®eld,87 and the Parm98.dat parameter set88 for our simulation, because it has been shown to perform well in the simulation of proteins and nucleic acids.88 ± 91 Molecular dynamics simulations have been shown in recent years to be important tools for elucidating structural, dynamic, and thermodynamic features of macromolecular systems.90,92,93

Results T7DNAP accommodates the BPDE-modified guanine base in the syn conformation Although the high-resolution NMR solution structure of (‡)-ta-[BP]G in duplex DNA showed that the modi®ed guanine base was in the normal B-DNA anti conformation, with the BP moiety in the minor groove and all base-pairs intact,74,76 the high-resolution NMR solution structure of this adduct at a model primer-template junction revealed that the modi®ed guanine residue existed in the syn conformation.77 The region of the T7DNAP protein in contact with the minor groove side of the primer-template junction, where the BP moiety would reside if the modi®ed guanine base adopted the anti conformation, is very densely

Carcinogen-induced Mismatch in a DNA Polymerase

packed, and there is no room to accommodate the bulky BP moiety. In addition, it has been shown experimentally, that the polymerase protein scans the minor groove side of the primer-template complex for shape anomalies that would indicate a mismatch in the newly formed base-pairs.1,32,42,43,45 In order to place the BP moiety in a favorable position, and avoid disrupting important interactions between the polymerase and the DNA, we modeled the (‡)-ta-[BP]G residue in the syn conformation, placing the carcinogen on the major groove side of the primer-template. Figure 2(a) shows the modeled starting structure of the T7DNAP/DNA complex, including the polymerase and thioredoxin proteins, BP-modi®ed template, primer, incoming dATP, and catalytic Mg2‡. The syn conformation of the (‡)-ta-[BP]G, which places the bulky aromatic BP moiety in a sterically feasible position in the major groove within an open channel of the protein, avoids disruption of the protein-DNA interactions. This conformation yields a (‡)-ta-[BP]G:A mismatched base-pair that closely resembles the size and shape of a normal Watson-Crick base-pair (discussed below). A structural transition occurs after 170 ps of molecular dyanamics After modeling the DNA, with the (‡)-ta-[BP]G in the syn conformation, into T7DNAP, minimizing, and equilibrating, as described in Materials and Methods, 1 ns of unrestrained MD was performed using the SANDER module of AMBER 5.0. Figures 2(b) and 3 show the entire structure and active site, respectively, of the T7DNAP/DNA structure after 1 ns of MD. Figure 4 shows an allatom, root-mean-square deviation plot for the solute over the complete MD simulation. The average all-atom RMSD for the entire structure was Ê throughout the simulation; this aver2.58(0.35) A age includes all atoms of the DNA and protein, including ¯exible loop regions. The simulation system consisted of 111,326 atoms, extremely large by current standards. This large size precludes longer simulation, but we feel that the 1 ns simulation was suf®cient due to the little overall motion of the system. The average RMSD of the DNA, including the three base 50 template overhang and the incomÊ . The DNA in the ing dATP, was 1.83(0.35) A active site adopts a conformation with characteristics intermediate between A and B-form DNA, similar to that observed in the crystal structures of T7 DNA polymerase4 and other DNA polymerases.26,29,30,94,95 The backbone torsion angles of the DNA in the active site of both the modeled structure and the T7 DNA polymerase crystal structure are listed in Table S8 of the Supplementary Material. Torsion angle d is near 80  in canonical A-form DNA, characteristic of C30 -endo sugar pucker, while it is near 150  in the canonical Bform DNA, characteristic of C20 -endo sugar pucker.96 The d angle of the DNA residues in the

Carcinogen-induced Mismatch in a DNA Polymerase

523

Figure 2. (a) Stereoview of the starting structure for the simulation after the 250 ps of equilibration. Color code: pink, polymerase/thioredoxin protein (ribbon representation); cyan, template DNA strand; yellow, modi®ed guanine base; purple, BP moiety; green, incoming dATP; gray, primer DNA strand; and royal blue, catalytic Mg2‡. (b) Stereoview of the simulated structure after 1 ns of unrestrained molecular dynamics; same color scheme as in (a). Figures are prepared for viewing with a steroviewer.

active site of our simulation was mainly near the C30 -endo A-type conformational region.

Figure 3. Active site of the simulated system after 1 ns of unrestrained molecular dynamics. See the legend to Figure 2 for color code.

A two-dimensional RMSD analysis of the active site (18 residues) of the modeled structure, which shows the RMSD agreement over time of each structure to every other structure and includes Ê of the nascent base-pair, every residue within 5 A revealed that the active site underwent a transition after approximately 170 ps, then remained stable for the rest of the simulation (Figure 5(a)). The active-site region had an average RMSD of Ê , and appears to exist in two states, 0.89(0.10) A one state prior to 170 ps and another after 170 ps, which persisted for the remainder of the simulation. Figure 5(b) shows that the (‡)-ta-[BP]G residue also underwent a transition after 170 ps of unrestrained MD, as demonstrated by the two distinct blue squares along the plot's diagonal; the second state was extremely stable from 170 ps until the end of the simulation. The (‡)-ta-[BP]G Ê. residue had an average RMSD of 0.81(0.17) A The transition in the position of the modi®ed guanine was primarily comprised of a shift in the position of the BP moiety with respect to the nascent

524

Carcinogen-induced Mismatch in a DNA Polymerase

Figure 4. All-atom RMSD of the protein/DNA system with respect to time.

base-pair, as shown by the comparison of the BP moiety position in Figure 2(a) and (b). In order to better understand the transition in the active site at 170 ps, we monitored the w, a0 , and b0 torsion angles (see Figure 1(a)) of the (‡)-ta-[BP]G residue with respect to time, as shown in Figure 6. This analysis shows the transition in the BP moiety that shifts the bulky carcinogen so that it contacts with polymerase surface residues, shielding one of its aromatic faces from contact with the solvent by extruding water (observed in trajectory visualization). This is apparent in Figure 3, which clearly shows close contact

between the BP moiety and protein residues. The glycosidic torsion angle, w, remains fairly constant throughout the simulation, changing only slightly from an average of 47.9  prior to 170 ps to 50.7  after 170 ps. However, the torsion angles governing the position of the BP moiety, a0 and b0 , change more signi®cantly at 170 ps. The a0 torsion angle average averages ÿ150.1  before 170 ps and ÿ138.4  after the transition at 170 ps; and the b0 torsion angle averages ÿ77.4  before 170 ps and ÿ65.1  following 170 ps. These differences in a0 and b0 after 170 ps re¯ect the change in the position of the BP moiety with respect to the modi®ed

Figure 5. (a) Pairwise plot of the RMSD of the polymerase active site, including all atoms of every residue (18 Ê of the (‡)-ta-[BP]G or the incoming dATP, with respect to time over 1 ns of unrestrained total) that comes within 5 A molecular dynamics. (b) Pairwise plot of the RMSD of the (‡)-ta-[BP]G residue with respect to time over 1 ns of unrestrained molecular dynamics.

525

Carcinogen-induced Mismatch in a DNA Polymerase

Figure 6. Fluctuation of the torsion angles of the (‡)-ta-[BP]G residue over the 1 ns of unrestrained molecular dynamics. Color code: green, w; blue, b0 ; and red, a0 . De®nitions of w, a0 , and b0 can be found in the legend to Figure 1(a).

guanine base; the carcinogen rotates towards the 50 side of the modi®ed template DNA strand to contact a number of amino acid residues, including Thr523, Ile540, Gln539, and Lys536, burying one of its hydrophobic planar faces from contact with the solvent (see Figure 2). The conformation adopted by the (‡)-ta-[BP]G residue after 170 ps appears stable on at least the 1 ns timescale, showing how the BP moiety and modi®ed guanine base may be accommodated stably in the active site of the polymerase opposite the mismatched dATP. The width of the nascent base-pair within the active site is similar to that of a normal Watson-Crick base-pair As discussed in the Introduction, DNA polymerases employ geometric selection in order to ensure that the nascent base-pair is matched correctly. One characteristic that is important during replication is the width of the nascent base-pair, measured as the distance between the C10 of the template base and that of the incoming nucleotide. An optimized A-T Watson Crick base-pair has a Ê ,40 and the distance C10 to C10 distance of 10.8 A 0 between the C1 of the template base and that of the incoming nucleotide in the original crystal structure of the T7DNAP ternary complex was Ê . Computational and experimental studies 10.5 A have suggested that DNA polymerases can accommodate base-pairs with C10 to C10 distances of up Ê .38,40 The ef®ciency of incorporation drops to 12 A signi®cantly when the C10 to C10 distance is greater Ê .38,40 Therefore, we measured the disthan 12.5 A

tance between the C10 of the (‡)-ta-[BP]G residue and that of the incoming dATP in our simulation to determine if the mismatch is within the size of nascent base-pairs that has been determined to be accommodated readily by DNA polymerases: the results are shown in Figure 7(a). Over the 1 ns simulation, the average C10 to C10 distance between the modi®ed guanine and the incoming Ê , which is within the nucleotide was 12.09(0.27) A range where incorporation is observed. While incorporation ef®ciency may be reduced, this is consistent with the infrequent, but biologically relevant mismatch of adenine with the (‡)-ta-[BP]G adduct during DNA replication bypass by T7 DNA polymerase (R.A.P., O. Rechkoblit, N.E. Geacintov & D.A. Scicchitano, unpublished results).61 The primer is poised to attack the incoming nucleotide The crystal structure of the T7DNAP ternary complex contained dideoxynucleotides at both the primer terminus and the incoming nucleotide.4 However, we modeled 30 hydroxyl groups on both the 30 -terminal base of the primer (A11) and the incoming dATP to simulate more accurately a replication complex in which nucleotide incorporation was about to occur (see Materials and Methods). During nucleotide incorporation, the Pa of the incoming deoxynucleoside triphosphate (dATP in this model) undergoes a nucleophilic attack by the 30 oxygen atom of the 30 -terminal base of the primer DNA strand (A11 in this model,

526

Carcinogen-induced Mismatch in a DNA Polymerase

Figure 7. (a) The distance between the (‡)-ta-[BP]G C10 and dATP C10 over the 1 ns unrestrained molecular dynamics simulation, measuring the width of the nascent base-pair. (b) The distance between the A11 O30 (30 -terminal oxygen atom of the primer) and the Pa of the incoming dATP over the 1 ns of unrestrained molecular dynamics.

see Figure 1(b)).1,3,23,97 In order for the reaction to proceed, and the nucleophilic attack to take place, the Pa of the incoming nucleotide must be adjacent to the nucleophilic 30 -OH of the primer strand. To determine whether the nascent base-pair was in the proper position for the formation of the phosphodiester bond within our modeled T7DNAP complex, we monitored the interatomic distance between the Pa of the incoming dATP and the would-be nucleophile, O30 of the 30 -terminal adenine base (A11) on the primer strand (Figure 7(b)). The average distance between the Pa of dATP and Ê . This is approximately O30 of A11 is 3.13(0.12) A equal to the sum of the van der Waals radii of oxygen and phosphorus.98 Therefore, the Pa of dATP

and O30 of A11 are in close proximity during our simulation, indicating that the nascent base-pair is in the proper position for the formation of the phosphodiester bond that occurs during nucleotide incorporation. Hydrogen bonds stabilize the nascent base-pair Analysis of the hydrogen bonds stabilizing the nascent base-pair lends insight into interactions important in maintaining the proper orientation of the active-site constituents. Table 1 lists the hydrogen bonds involving the incoming dATP and/or (‡)-ta-[BP]G residues that are intact for greater

527

Carcinogen-induced Mismatch in a DNA Polymerase

than 30 % of the simulation and those involving the hydroxyl groups of the BP moiety that are intact for greater than 10 % of the simulation. The incoming dATP is involved in eight hydrogen bonds with the protein, one that was occupied for greater than 30 % of the simulation, while the remaining seven were occupied for between 64 % and 95 % of the simulation. Two interactions between the protein and dATP O30 are present in the simulation that were not seen in the crystal structure due to the fact that the crystal structure employed a dideoxynucleotide as the incomer. The dATP O30 was the acceptor for a hydrogen bond with both main-chain Glu480 HNa (occupied for 80.5 % of the simulation) and Tyr526 HZ (occupied for 92.0 % of the simulation), which further stabilized the position of the incoming nucleotide in comparison to that seen in the X-ray crystal structure.4 All of the interactions between the protein and incoming nucleotide deemed important for catalysis and ®delity in the crystal structure4 were maintained throughout the simulation, including those with Glu480, His506, Arg518, Lys522, and Tyr526. The (‡)-ta-[BP]G residue was also involved in multiple hydrogen bonding interactions; those identi®ed in the crystal structure involving the unmodi®ed template base were maintained throughout our simulation. In addition, new interactions were identi®ed that resulted from the abnormal syn conformation of the modi®ed guanine base and the major groove position of the BP moiety. Signi®cantly, O6 of the modi®ed guanine base and incoming dATP H6 were involved in a hydrogen bond for 63.9 % of the simulation, with an average heavy-atom to heavy-atom disÊ and an average hydrogen tance of 3.04(0.31) A bonding angle of 169.3(4.80)  over the entire

simulation. Furthermore, as seen in Figure 8, this hydrogen bond remains stably in place following 550 ps. The (‡)-ta-[BP]G H1 was involved in a hydrogen bond with Thr523 Og1 for 59.2 % of the simulation. In addition to positioning the H1 and O6 atoms of the modi®ed guanine base in proper positions to form hydrogen bonds with the protein and incoming dATP, respectively, the syn conformation of the modi®ed guanine base placed the hydroxyl groups of the BP moiety in close proximity to polymerase residues with which they were able to form hydrogen bonds, as shown in Table 1. The O8 atom of the BP moiety was involved in three hydrogen bonds with Lys536, each for more than 10 % of the simulation, and the HO7 atom of the BP moiety was involved in a hydrogen bond with Gln533 for 21.5 % of the simulation. The position of the carcinogen was stabilized by these hydrogen bonding interactions, in addition to the previously mentioned hydrophobic interactions of the BP moiety with Thr523, Ile540, Gln539, and Lys536. The stabilizing hydrogen bonds and hydrophobic interactions of the modi®ed base and carcinogen complement the fact that the syn conformation of the (‡)-ta-[BP]G residue and presence of the BP moiety on the major groove side of the DNA within the polymerase active site do not interfere with interactions deemed important in the ®delity and processivity of T7DNAP.1,4 The aforementioned hydrogen bonds, involving the modi®ed guanine base and carcinogen moiety, all on the major groove side of the DNA, were a direct result of the (‡)-ta-[BP]G residue adopting the syn, as opposed to the normal anti, conformation within the active site and the BP moiety residing on the major groove side of the DNA, within an open pocket of the polymerase protein.

Table 1. Hydrogen bonds involving the (‡)-ta-[BP]G or dATP residues Donor mcGly533 HNa [BP]G H6 Lys536 Hz1 Lys536 Hz2 Lys536 Hz3 [BP]G HO7 dATP H61 mcGly479 HNa His506 HNe2 Arg518 HZ22 Arg518 HZ12 Lys522 Hz2 Lys522 Hz3 Tyr526 HZ A11 HO30 T19 H3 dATP HO30 mcGlu480 HNa

Acceptor

% Occupied

Mean length heavy to Ê) heavy atom (A

Mean angle (deg.)

Identified in crystal structure?

[BP]G OP Thr523 Og1 [BP]G O8 [BP]G O8 [BP]G O8 Gln539 Oe1 [BP]G O6 dATP Og2 dATP Ob1 dATP Og2 dATP Og3 dATP Oa1 dATP Og3 dATP O30 dATP O50 dATP N1 dATP Ob1 dATP O30

69.7 59.2 11.7 13.2 11.5 21.5 63.9 32.1 80.4 89.0 94.7 70.5 64.4 92.0 67.1 34.3 99.8 80.5

2.91 0.13 3.00  0.13 2.87  0.11 2.91  0.12 2.88  0.12 2.79  0.16 2.91  0.13 3.16  0.11 2.86  0.11 2.76  0.09 2.72  0.07 2.85  0.11 2.77  0.10 2.67  0.10 2.82  0.13 3.02  0.12 2.54  0.08 3.06  0.12

168.80  4.84 167.77  4.80 168.83  4.8 168.89  5.3 168.84  5.12 168.57  4.82 169.28  4.80 168.90  4.67 168.02  4.49 168.51  4.33 170.81  4.52 168.88  4.79 168.11  4.64 171.32  4.56 168.89  4.77 167.97  4.72 172.93  3.53 170.43  4.59

Yes No No No No No No No Yes Yes Yes Yes No No No No No No

% Occupied indicates the percentage of the time during the 1 ns unrestrained molecular dynamics simulation that the hydrogen Ê between heavy atoms and a hydrogen bonding angle of 160  . bond was intact. Hydrogen bond criteria were 3.3 A

528

Carcinogen-induced Mismatch in a DNA Polymerase

Figure 8. Hydrogen bond distance between (‡)-ta-[BP]G O6 and dATP N6 as a function of time.

Discussion The nascent base-pair is stably accommodated within the polymerase active site with the BP moiety on the major groove side of the DNA Replicative DNA polymerases employ several mechanisms for ensuring the faithful replication of the genome, including geometric selection to discriminate against mismatched incoming nucleotides in the active site.36 ± 38,41 Despite these proofreading mechanisms, carcinogen exposure increases the mutation frequency because carcinogen-DNA adducts are misreplicated by DNA polymerase proteins more frequently.12 ± 19,21,49 We carried out a molecular dynamics simulation of T7DNAP containing a (‡)-ta-BPDE-damaged template-primer complex and included a mismatched incoming dATP to elucidate structural features that allow this mismatch to evade the proofreading machinery of the polymerase protein, resulting in a mutation. We conducted extensive equilibration of the system, followed by 1 ns of unrestrained molecular dynamics at constant pressure and temperature using the AMBER 5.0 molecular dynamics package.86 We modeled the (‡)-ta[BP]G residue in the syn conformation, with the BP moiety in the open pocket on the major groove side of the DNA, because we observed that the polymerase active-site was very densely packed on the minor groove side of the DNA and the BP moiety could not be accommodated in this region. During the simulation, the entire protein-DNA complex was very stable, with only a small readjustment of the (‡)-ta-[BP]G residue and active-site region after 170 ps of unrestrained MD. The BP moiety rotated towards the 50 end of the template

to interact with several residues in the polymerase, burying one side of the hydrophobic aromatic moiety from the solvent. The transition of the (‡)-ta[BP]G residue and the active-site region show that the nascent base-pair and the adjacent protein residues rearranged to better accommodate the syn (‡)-ta-[BP]G:A mismatched base-pair and carcinogen moiety, after which they remained stable for the remainder of the simulation. The proper ®t of the syn (‡)-ta-[BP]G:dATP mismatch within the active site of T7DNAP was also demonstrated by the width of the base-pair during our simulation, as judged by the distance between (‡)-ta-[BP]G C10 and dATP C10 during our simulation, which was within the limits of that known to be accommodated by DNA polymerases.38,40 In addition, monitoring the distance between the 30 -OH group of the primer terminus and the Pa of the incoming dATP showed that these atoms were in close proximity throughout the simulation, a requirement for the catalysis by T7DNAP, in which the primer terminus 30 -OH attacks the Pa of the incoming nucleotide to form a phosphodiester bond. Hydrogen bonding analysis of the simulation system also revealed interactions that further stabilized the (‡)-ta-[BP]G:dATP mismatch. A hydrogen bond between the syn (‡)-ta-[BP]G and incoming dATP was present during the simulation. This indicated that the mismatch had adopted a conformation that minimized steric clashes, and optimized hydrogen bonding interactions between the nascent base-pair. Hydrogen bonds between the (‡)-ta-[BP]G and the polymerase protein were present for the majority of the simulation, including those observed in the original crystal structure between the template base and protein, and novel

Carcinogen-induced Mismatch in a DNA Polymerase

hydrogen bonds that formed as a result of the syn conformation of the (‡)-ta-[BP]G and the presence of the BP moiety on the major groove side of the DNA. The incoming dATP was stabilized by a number of hydrogen bonds with the polymerase protein, corresponding almost exactly with those observed in the crystal structure of the T7DNAP ternary complex.4 In addition, new hydrogen bond interactions were identi®ed between the 30 -OH group of the incoming dATP and the protein compared to the original crystal structure, in which the incoming nucleotide was ddGTP, lacking a 30 -OH group.4 Blocking anti conformation versus the readthrough syn conformation of the modified guanine base Experimental evidence shows that BPDEmodi®ed guanine primarily blocks replication by T7DNAP;61,69,70 occasionally, however, the polymerase replicates past the adduct, during which it inserts the incorrect base more frequently than during normal replication of undamaged DNA (R.A.P., O. Rechkoblit, N.E. Geacintov & D.A. Scicchitano, unpublished results).61 We hypothesize that the normal anti conformation of (‡)-ta-[BP]G blocks replication by T7DNAP because the steric bulk of the BP moiety on the DNA minor groove side prevents the pairing of the template base with an incoming nucleotide and inhibits the normal conformational change of the enzyme from the open to the catalytically active closed conformation. Data obtained by Alekseyev & Romano99 appear to be consistent with the alteration of this conformational transition in E. coli DNA polymerase I by the presence of a (‡)-ta-[BP]G adduct. However, the (‡)-ta-[BP]G adduct is occasionally bypassed during processive replication, and our simulation suggests that (‡)-ta-[BP]G adopts the syn conformation in the active site during bypass by replicative DNA polymerases, allowing the modi®ed guanine base to pair with an incoming deoxynucleoside triphosphate and the polymerase to adopt the closed conformation required for the formation of the phosphodiester bond. The syn (‡)-ta-[BP]G places the BP moiety on the major groove side of the DNA, in an open cavity of the polymerase protein, avoiding steric interference of residues experimentally found to be important in the replicative and ®delity-ensuring function of T7DNAP.1,4 In addition, the syn (‡)-ta-[BP]G:dATP mismatch closely resembles the size and shape of a normal Watson-Crick base-pair (see Figure 9), which could trick the geometric selection machinery37 ± 39,41 of the polymerase active site into accepting the mismatch and proceeding with the formation of the phosphodiester bond and extension of the primer. We hypothesize that bypass involving other dNTP incoming nucleotides, including even dCTP, also involves the syn conformation of the (‡)-ta-[BP]G template base, and this issue will be investigated in further studies.

529 The present work does not rule out other conformations for the bypass of this adduct during replication. The presence of multiple differing conformations for the (‡)-ta-[BP]G adduct has been observed in high-resolution NMR studies,17,74 ± 78 in computational studies,100 ± 102 and has been suggested in mutagenesis studies;16,102,103 sequence effects also play an important role here.13,16,67,103,104 However, the present work suggests a plausible informational structure for the rare mutagenic event in which the BPDE lesion is bypassed by a replicative DNA polymerase, with the misincorporation of an adenine residue. The bypass of a (‡)-ta-[BP]G template base in the anti conformation, with the BP moiety on the densely packed minor groove side of the DNA, would likely require major rearrangements of polymerase residues critical to the function and ®delity of the processive enzyme. However, recent ®ndings have implicated various bypass polymerases in enabling DNA synthesis past a number of lesions.105 ± 108 Indeed, Fuchs and co-workers21 found that the induction of G-to-T transversions by the (‡)-ta[BP]G adduct is independent of SOS-encoded functions in E. coli, and hence is probably carried out by the replicative DNA polymerase III holoenzyme. On the other hand, these workers ®nd that error-free replication and the production of frameshift mutations are dependent on SOS induction of DNA polymerases specialized in translesion synthesis (pol IV and pol V). The ®delity machinery of such bypass polymerases may operate differently from the high-®delity replicative DNA polymerases, like T7 and pol III, and are currently the subject of active investigation.

Figure 9. Comparison of the shape of the syn (‡)-ta[BP]G:dATP base-pair (from 1 ns structure) with that of a normal anti-T:dATP Watson-Crick base-pair. Color code: yellow, modi®ed guanine base; purple, BP moiety; green, dATP; black, hypothetical thymine; red line, hydrogen bond between (‡)-ta-[BP]G O6 and dATP HN6 present during the simulation; blue lines, hydrogen bonds between dATP and hypothetical anti thymine.

530

Conclusion Our simulation provides an atomic-resolution structural and dynamic view of a replicative DNA polymerase poised to render an environmental carcinogen-DNA adduct into a mutation. The simulation of T7DNAP containing a BPDE-modi®ed template primer and mismatched dATP shows how this mismatch can be stabilized, with the modi®ed guanine base adopting the syn conformation and the carcinogen moiety residing on the major groove side of the DNA, in an open pocket of the polymerase. The high-resolution NMR solution structure of (‡)-ta-[BP]G at a model replication fork, with no partner nucleotide opposite the adduct, has shown the modi®ed guanine base in a syn conformation.77 Our simulation suggests that a syn conformation of (‡)-ta-[BP]G, with the BP moiety on the major groove side of the DNA, could allow bypass replication of the adduct. The BP moiety in the major groove does not disrupt interactions between the template base and the protein, nor does it sterically impede the binding of an incoming nucleotide. An anti conformation places the BP moiety on the minor groove side of the DNA, which is densely packed with polymerase residues important to function and ®delity.1,4 On the minor groove side of the DNA, the BP moiety would obstruct the polymerase active site and could inhibit the polymerase closing motion needed for catalysis.99 Thus, the anti conformation could account for the predominant biological consequence of the (‡)-ta-[BP]G adduct, namely replication blockage. On the other hand, the rare and infrequent bypass of the lesion during replication61,69,70 (R.A.P., O. Rechkoblit, N. E. Geacintov & D. A. Scicchitano, unpublished results) may result from the syn conformation of the adduct, as seen in our simulation. We propose that adenine is misincorporated most frequently opposite the damaged guanine base because its size and shape opposite syn (‡)-ta-[BP]G is similar to that of a Watson-Crick base-pair. Thus, our simulation lends insight into the mechanism by which bulky DNA adducts cause mutations during DNA replication. In future work, we hope to examine the relative binding free energies of the (‡)-ta-[BP]G adduct/polymerase system containing each of the other three incoming nucleotides using the recently developed MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method.93,109 ± 114

Carcinogen-induced Mismatch in a DNA Polymerase Ê cutoff was applied to Lennardout the simulation. A 9 A Jones interactions, the non-bonded interaction list was updated every step, periodic boundary conditions were applied, and all molecular dynamics runs were carried out at constant pressure and temperature (Berendsen temperature coupling), unless indicated otherwise. Parameterization Partial charges We obtained partial charges from a number of different conformations in order to best minimize conformational bias. For parameterization of the (‡)-ta-[BP]G, single-stranded 5-methyl cytosine (5meCS), and 50 -terminal 5-methyl cytosine (5meCT) residues, 12 different conformations were obtained from energy minimization studies (R.A.P. & S.B., unpublished results) of a singlestrand/double-strand junction at the (‡)-ta-[BP]-modi®ed p53 hotspot 157 sequence. These structures included syn and anti conformations of the modi®ed guanine base, and A and B-form helical conformations of the double-stranded region, as well as 12 different combinations of a0 and b0 , the torsions governing the orientation of the BP moiety (Figure 1(a)) with respect to the modi®ed guanine (Supplementary Material, Table S7). Partial charges for the double-stranded 5-methyl cytosine (5meCD) were obtained from the double-stranded 5meC within the lowest energy B-form single-stranded/ double-stranded junction from the energy minimization studies. The incoming ddGTP in the T7DNAP crystal structure was remodeled to dATP using the Builder module of InsightII (Molecular Simulations, Inc.), including the addition of a 30 hydroxyl group, and the resulting dATP molecule was used for calculations. Hartree-Fock calculations with basis set 6-31G* in GAUSSIAN 94 were used to calculate the partial charge of each atom in all ®ve residues parameterized.117 The RESP module of AMBER 5.0 was then used to ®t the charge to each atomic center in the molecule. Charges were then averaged, where more then one conformation was used, and normalized to avoid charge imbalance in each residue (Wu et al.,118 see the Appendix). (The ®nal partial charges and AMBER atom type assignments are shown in the Supplementary Material, Tables S1-S5). Bond angle and improper torsion parameters Table S6 (Supplementary Material) lists the bond angle and improper torsion parameters that were assigned by analogy to chemically similar types in the AMBER database Parm98.dat parameter set.88 Mg2‡ parameters Parameters for the magnesium ions were adapted from values obtained by J. Aqvist,119 with an R* of Ê and an e of 0.8749 kcal molÿ1. 0.7926 A

Materials and Methods

Preparation of the starting structure

Simulations were carried out using the SANDER module of AMBER 5.086,115 and the Cornell et al. force-®eld,87 including the Parm98.dat parameter set.88 The particle mesh Ewald method116 was used to approximate the important coulombic interactions, the SHAKE option was used to constrain bonds involving protons (tolerÊ ), and a 2 fs time-step was used throughance ˆ 0.0005 A

Ê resolution coordinates of the closed conforThe 2.2 A mation of the T7DNAP/thioredoxin complex were obtained from the Protein Data Bank120 (PDB ID: 1T7P).4 The crystal structure included most of the coordinates of the polymerase and thioredoxin proteins, primer and template DNA molecules, incoming deoxynucleoside triphosphate, three (out of four) catalytic Mg2‡, and 503

531

Carcinogen-induced Mismatch in a DNA Polymerase water oxygen atoms. The hydrogen atoms of the water molecules were added using the Builder module of InsightII (Molecular Simulations Inc.). Missing residues (polymerase residues 166, 189, 285, 293, and 571, and thioredoxin residues 1,2, and 108), loops (polymerase residues 293-318 and 576-586), and the hydrogen atoms of the polymerase and thioredoxin proteins, unable to be located in the crystal structure, were modeled using the LOOK (Molecular Applications Group) homology modeling program by Dr Suresh Singh (Molecular Systems, Merck Research Laboratories). In addition, we modeled the missing Mg2‡ in the exonuclease domain according to its expected position, as described by Doublie et al.,4 and then minimized the Mg2‡ in the exonuclease domain for 1000 steps of steepest descent (SD) while holding the rest of the system ®xed. The DNA sequence was remodeled to match that of the p53 hotspot 157, including a dATP as the incoming nucleotide to simulate the incorporation of a mismatch, using the Builder module of InsightII. The crystal structure employed dideoxynucleotides at the 30 termini of the primer and template strands, as well as the incoming nucleotide.4 We modeled the 30 termini and incoming nucleotide as deoxynucleotides by adding 30 hydroxyl groups. In addition, the two most 50 bases of the template were modeled into a canonical B-form conformation, since these two end nucleotides were not located in the original crystal structure. The sequence of the modeled DNA is shown in Figure 1(b). The modeled protons were minimized for 100 steps of SD followed by 400 steps of conjugate gradient (CG) minimization using the Sander module of AMBER 5.0 and the Hingerty distance-dependent dielectric function (implemented in our version of AMBER 5.0).121 The modeled loops and residues were minimized for 1000 steps of SD followed by 4000 steps of CG. The modeled DNA sequence was minimized for 3000 steps of SD in order to alleviate steric strain, holding the phosphodiester backbone ®xed. The minimized DNA was then docked with the protein, and the DNA and all protein Ê of the DNA were minimized for residues within 12 A 1000 steps of SD and 4000 steps of CG while holding the rest of the protein ®xed. The minimized protein/DNA structure was then prepared for MD using the LEaP module of AMBER 5.0. First, the protein/DNA system was neutralized with 40 sodium ions, placed at the positions of minimum electrostatic potential of the system, and then the 503 crystallographic water molecules were added. The protein/DNA was held ®xed and the counterions and crystallographic water molecules were minimized for 5000 steps of SD to alleviate steric clashes. The neutral system was solvated Ê in 32,056 TIP3P water molecules,122 using a buffer of 9 A around the solute, creating a periodic box of dimensions Ê  100.19 A Ê  100.78 A Ê and a total of 111,326 130.42 A atoms. The solute (protein, DNA, and Mg2‡) was held ®xed and the Na‡ and water molecules were minimized for 5000 steps of SD prior to beginning equilibration. Molecular dynamics simulation and data analyses In order to relax the water molecules and ions, and bring the system to a realistic density of approximately 1 g cmÿ3, 50 ps of MD were conducted at 10 K whilst holding the solute ®xed with harmonic restraints of Ê ) (1 kcal ˆ 4.184 kJ). The system was uni25 kcal/(mol A formly heated to 300 K over 25 ps and then held at 300 K for 75 ps, again restraining the solute, in order to equilibrate the water molecules and ions. The remainder of the MD was carried out at 300 K. The restraints on the

solute were released slowly over 100 ps of MD at 300 K Ê) by running 10 ps of MD with 10.0 kcal/(mol A Ê ) restraints, and restraints, 40 ps with 1.0 kcal/(mol A Ê ) restraints on the solute, suc50 ps with 0.1 kcal/(mol A cessively, for a total equilibration time of 250 ps. Once the restraints on the solute were removed, unrestrained MD production runs were begun, with a total of 1 ns of unrestrained MD completed. The coordinates, velocities, and energies were collected every 500 fs in order to provide extensive details regarding the trajectory of the protein/DNA complex. The CARNAL and PTRAJ123 modules of AMBER 6.0 were used for the trajectory processing and analyses of the RMSDs, interatomic distances, hydrogen bonding, solvent density, and torsion angles throughout the simulation. The hydrogen bonding cutoff for heavy-atom to Ê and that for the hydroheavy-atom distance was 3.3 A gen bonding angle was 160  .

Acknowledgments We greatly appreciate the help of Dr Jerry Greenberg (NPACI/SDSC) throughout our work. We thank Dr Thomas Cheatham III (University of Utah) for providing us with the Ptraj module for water density analysis, much helpful advice in conducting the analysis, and critical reading of the manuscript. We also thank Dr Suresh Singh (Merck Research Laboratories) for help in modeling the loops and residues missing from the crystal structure. We thank Dr Robert Shapiro and Dr Nicholas E. Geacintov (New York University) for ongoing helpful discussions. Computations were carried out at NSF NPACI facilities at the San Diego Supercomputer Center and the University of Texas at Austin and the DOE National Energy Research Supercomputer Center; the help of the staff at these facilities is greatly appreciated. The AMBER re¯ector, [email protected], was also of great assistance. Support for this work through NIH grant CA28038 and DOE grant DE-FG0290ER60931 is gratefully acknowledged.

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Edited by B. Honig (Received 14 December 2000; received in revised form 26 March 2001; accepted 26 March 2001)

http://www.academicpress.com/jmb Supplementary Material for this paper, comprising eight Tables is available from IDEAL