Increased Flexibility Enhances Misincorporation

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 °C and results compared with an earlier study at 37 °C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 °C compared with 37 °C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 °C compared with 37 °C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

ensuring faithful replication of DNA, resulting in higher error rates during replication of undamaged DNA templates and a greater ability to bypass certain aberrant bases. Dpo4 from the crenarchaeon Sulfolobus solfataricus P2 offers a valuable opportunity to investigate the effects of conformational flexibility on carcinogen-DNA adduct bypass by Y-family DNA polymerases because of its thermal stability and ability to function efficiently at a wide range of temperatures (10).
Unlike high fidelity replicative DNA polymerases, Dpo4 does not employ an induced-fit mechanism for selecting the correct nucleotide (7,11). It has a more flexible active site than replicative DNA polymerases (6,7,(12)(13)(14)(15)(16)(17) and can thus accommodate base pairs of different sizes (7,18). Dpo4 and other Y-family DNA polymerases bind the substrate with fewer interactions than do high fidelity replicative enzymes (12, 13, 15, 16, 19 -25); this leaves both the minor and major groove sides of the nascent base pair solvent exposed (7), but with more open space on the major groove side. A crystal structure has revealed that a bulky DNA adduct derived from benzo[a]pyrene (BP) 4 can be accommodated in this spacious pocket (26). The limited protein-DNA interactions and relaxed fidelity may help explain why Dpo4 is distributive, relatively error-prone when replicating undamaged DNA (10,27), and able to bypass some bulky DNA adducts more readily than high fidelity enzymes (10,26,28,29).
We show here that an increase in temperature from 37 to 55°C increases bypass of the (ϩ)-ta-[BP]G adduct by Dpo4, particularly due to enhancement of incorrect nucleotide insertion opposite the lesion. Molecular modeling and molecular dynamics simulations show that the BP rings can be accommodated in the active site of Dpo4 in the smaller minor groove-side space (28,58) or the larger major groove-side pocket, with the modified guanine adopting the anti or syn glycosidic torsion conformation, respectively, at both 37 and 55°C. However, at 55°C the major groove/syn orientation is structurally less distorted and more favorable for mismatch incorporation; the minor groove/anti conformation, permitting near Watson-Crick pairing, remains preferred for the normal partner dCTP. The molecular dynamics simulations are consistent with the k cat values for nucleotide incorporation opposite the lesion, and structurally explain the experimental findings. The results further suggest that the anti/syn equilibrium is temperature-dependent and influences the overall rate of catalysis, enzyme fidelity, and specificity.

EXPERIMENTAL PROCEDURES
Modified Oligodeoxynucleotide Template and Primer Strands-The construction of BP diol epoxide-modified oligonucleotide strands was carried out as reported previously (28). In brief, the 11-mer oligonucleotide, 5Ј-TTATAG 6 *CACAC-3Ј with the single (ϩ)-ta-[BP]G adduct at G 6* , was generated by a direct synthetic method, purified, and characterized as described previously (59 -62). The modified 11-mers and flanking 12-and 19-mers were annealed with a complementary 38-mer template strand (all purchased from Sigma Genosys), PAGE-purified, and then the three oligonucleotides were ligated using T4 DNA ligase (New England BioLabs, Inc., Beverly, MA) as described (28,30,32,60). The resultant 42-mer modified oligonucleotides (5Ј-CACGTAATGATGTTATAG 6 * CACACGCTATCTGGCCAGATCGCG-3Ј) were purified using 20% polyacrylamide gel electrophoresis containing 8 M urea. An unmodified control 42-mer template strand was con-structed in a similar manner. The purity of the template was verified by end labeling with T4 polynucleotide kinase (New England BioLabs, Inc., Beverly, MA) and denaturing PAGE. The integrity of the modified oligonucleotide was verified by observing that the electrophoretic mobility of the 42-mer strand containing the (ϩ)-ta-[BP]dG adduct located at the 25th nucleotide was somewhat slower than that of the unmodified 42-mer oligonucleotide. This difference is routinely utilized to monitor the integrity of a modified 42-mer sequence.
Primer-extension Assays-Primer-extension assays were carried out as reported previously (28). The primer strand utilized in the single dNTP incorporation experiments opposite the lesions (standing-start primer-extension assays) was 21 nucleotides in length and its 3Ј-terminal nucleotide extended up to the template base flanking the (ϩ)-ta-[BP]dG adduct on the 3Ј side (24th nucleotide from the 3Ј end of the template). In the running start primerextension assays, the primer strand was 18 bases in length and its 3Ј-terminal base extended up to the 21st template base counted from the 3Ј end. All template-primer complexes had a 3-base single strand overhang at the 3Ј side of the template. The primer strands were 5Ј-end labeled using 1 units of T4 polynucleotide kinase (New England BioLabs) and 0.01 mCi of [␥-32 P]ATP (3,000 Ci/mmol) (PerkinElmer Life Sciences) in 70 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , and 5 mM dithiothreitol. The labeled primers and unlabeled 42-mer DNA templates were annealed in a 1:1.25 ratio.
The running start primer-extension assays with Dpo4 were carried out in a 40-l volume containing 40 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , 10 mM dithiothreitol, 250 g/ml bovine serum albumin, 2.5% glycerol, 2000 M of each dNTP, and 10 nM Dpo4. These solutions were incubated at 37 or 55°C, 10-l aliquots were removed, and the reactions were terminated by adding 10 l of a "stop solution" (80% formamide, 5 mM Tris boric acid, 1 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue) after 15, 30, and 60 min. Standing start primer-extension assays were utilized to determine the k cat for insertion of each nucleotide by Dpo4 and were carried out with only one dNTP at a time. The actual measurements yielded the Michaelis-Menten parameters V max , the rate of dNTP incorporation at saturating concentrations of each dNTP, as described earlier (33), and the relationship k cat ϭ V max /[E 0 ] was utilized to estimate k cat , where [E 0 ] is the enzyme concentration. The 40-l reaction solutions contained 40 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , 10 mM dithiothreitol, 250 g/ml bovine serum albumin, 2.5% glycerol, 2.5 nM [ 32 P]primer-template, and 10 nM Dpo4. The concentration of dNTP and reaction times were dependent on the saturation conditions for each dNTP. The dNTP concentrations and reaction times utilized are provided in supple- mental Table S1. V max values are given in supplemental Table  S2. All reaction stop mixtures were heated to 90°C for 10 min prior to analysis on a 20% PAGE gel containing 8 M urea. The gels were dried and exposed to a PhosphorImager plate prior to data analysis with ImageQuant software (Amersham Biosciences). All assays were conducted in triplicate (three independent trials) and the results are reported as averages with standard deviations.
Molecular Modeling and Molecular Dynamics Simulations of Dpo4 Ternary Complexes Containing the (ϩ)-ta-[BP]G Adduct-Parameters for the (ϩ)-ta-[BP]G, dNTP, and Mg 2ϩ residues developed previously were employed in the current work (28,(63)(64)(65). All molecular modeling and molecular dynamics simulations were carried out as described in Perlow-Poehnelt et al. (28) except that the systems were heated to and maintained at 328 K (55°C) instead of 310 K (37°C). In brief, the type I crystal structure of the Dpo4 ternary complex (7) was used as the starting structure for the molecular models (Protein Data Bank code 1JX4) with coordinates obtained from the Protein Data Bank (66). The primer-template sequence used in the simulations is shown in Fig. 1b. Both the anti and syn conformations of the template (ϩ)-ta-[BP]G were employed and each anti dNTP was modeled opposite the adduct. Systems were also constructed in which (ϩ)-ta-[BP]G in the anti conformation was modeled opposite syn dATP and syn dGTP. A templating base and an incoming dNTP in the syn conformation have been observed in pol (67) and Dpo4 (29), respectively. Therefore, a total of 11 simulations was carried out, including an unmodified control. The starting torsion angles for the anti and syn (ϩ)-ta-[BP]G residues (see Fig. 1a) were: , 236°; ␣Ј, 109°; ␤Ј, 248°and , 71°; ␣Ј, 321°; ␤Ј, 230°, respectively. These combinations of , ␣Ј, and ␤Ј are within domains computed to be favorable for this adduct (68). The anti and syn dNTPs had starting torsions of 222°and 42°, respectively. The 3Ј-OH groups were added to the dideoxynucleotides in the crystal structure, and hydrogen atoms were added to all residues using the LEaP module of AMBER 6.0 (69). Details of the protocols for the molecular dynamics simulations are identical to those given in Perlow-Poehnelt et al. (28), except that the production runs were carried out at 328 K (55°C).
Plots of root mean squared deviations demonstrating the stability of the simulations are shown in supplemental Fig. S1. Molecular alignments and Fig. 3 were produced using PyMol (Delano Scientific, CA).

Temperature Dependence of Translesion Bypass
The results of typical running start primer-extension experiments at different reaction times are depicted in Fig. 2a. Under the given experimental conditions, translesion bypass is limited at 37°C, but is significantly more pronounced at 55°C, particularly after 60 min. In all cases, primer-extension slows after incorporation of the nucleotide opposite the template base 3Ј to the damaged base, labeled as the Ϫ1 position in Fig. 2a. Insertion of the base opposite the adduct (position (0)) is the slowest, rate-determining step because partially extended primers accumulate just before the lesion. Extension from the lesion site appears to be significantly faster because there is little accumulation of primers with their 3Ј-terminal nucleotide opposite the (ϩ)-ta-[BP]G adduct. These results are consistent with those of Boudsocq et al. (10) who showed that Dpo4 bypasses several bulky adducts at least 1 order of magnitude more slowly than unmodified DNA. A noteworthy feature of the running start experiments is that insertion of the base opposite the lesion is slower than extension beyond the lesion once a nucleotide has been incorporated into the primer strand opposite the adduct. In contrast, in the case of replicative polymerases, primer-extension past the (ϩ)-ta-[BP]dG adduct is significantly slower than insertion of a base opposite the adduct (32,33,70). The extension step appears to pose a greater obstacle to replicative DNA polymerases, as well as in the case of some other Y-family polymerases (3,71,72). The running start experiments in Fig.  2a show that the rate-determining step in translesion bypass catalyzed by Dpo4 is the incorporation of a nucleotide opposite the (ϩ)-ta-[BP]dG adduct and justifies the focus of our modeling study on this particular step.
We next investigated the fidelity of nucleotide insertion utilizing the (ϩ)-ta-[BP]dG lesion as the template base. Typical standing start experiments are exemplified in Fig. 2b. In the case of unmodified templates, the non-mutagenic insertion of dC is dominant at both 37 and 55°C, but the mismatched nucleotides are also inserted to some extent; this is consistent with earlier results at 37°C (10). However, modification of the template base to (ϩ)-ta-[BP]dG changes the incorporation preference at both 37 and 55°C. At 37°C, all four nucleotides are inserted opposite (ϩ)-ta-[BP]dG, with dG insertion least favorable. However, at 55°C, all four nucleotides appear to be inserted opposite the adduct to similar extents (Fig. 2b).
The total amount of each nucleotide incorporated opposite the adduct is impacted by the rate constant of nucleotide incorporation, k cat , which is reflective of the slowest, rate-determining step in the reaction cycle. At 37°C, the k cat values for the insertion of all four dNTPs opposite (ϩ)-ta-[BP]G are small and comparable, ranging from ϳ7 to 11 ϫ 10 Ϫ4 min Ϫ1 (28). Increasing the reaction temperature to 55°C significantly enhances the rates of incorporation of the incorrect nucleotides opposite (ϩ)-ta-[BP]G by factors of 7.3, 8.0, and 9.6 for dT, dA, and dG incorporation, respectively. In contrast, k cat for dC incorporation is increased by a factor of only 2.5 (Fig. 2c, bottom  panel).

Overview of Modeling and Molecular Dynamics Simulations
To elucidate the structural underpinnings of the enhanced bypass and altered selectivity of mismatched nucleotide insertion opposite (ϩ)-ta-[BP]G at the higher temperature, molecular dynamics simulations were carried out at 55°C to compare with those previously conducted at 37°C (28). Both (ϩ)-ta-[BP]G and the incoming dNTPs were first modeled in their anti glycosidic conformations. We then evaluated whether the incoming purine dNTPs with a syn conformation opposite the anti damaged guanine are feasible. Finally, following our previous findings that syn (ϩ)-ta-[BP]G adduct conformers are more favorable for translesion bypass (28,(63)(64)(65), we investigated the feasibility of syn (ϩ)-ta-[BP]G:anti dNTP pairing within the active site of Dpo4. A total of 11 simulations was carried out, including an unmodified control DNA primer-template complex. These simulations were carried out at 55°C in parallel to those performed previously at 37°C (28). The same starting structures, parameters, and protocols were utilized to allow comparisons of the same ternary complexes at the two temperatures and to gain insights into possible origins of the effects of temperature on translesion bypass observed experimentally.

The Normal anti Conformation of (؉)-ta-[BP]G: Molecular Dynamic Simulations Indicate That the Conformations of the Nascent Base Pairs Are More Distorted at 55°C than at 37°C
anti dNTPs-The (ϩ)-ta-[BP]G adduct most often blocks replicative DNA polymerases (35,(63)(64)(65). This blockage is explained by an anti conformation of the (ϩ)-ta-[BP]G adduct, observed in a replicative polymerase crystal structure (35); in this case the bulky carcinogen ring system is on the crowded minor groove side of the nascent base pair, disrupting critical protein-DNA interactions. The anti conformation is also observed in double strand DNA (73). Our previous modeling studies showed that (ϩ)-ta-[BP]G can be accommodated in either the anti or the syn conformation, with the BP moiety either on the minor or major groove side, respectively, within the active site of Dpo4 (28). However, the anti conformation of the damaged guanine, with the BP moiety on the minor groove side, causes an opening of the cleft between the little finger, fingers, and palm domains of the enzyme. This is illustrated by comparing these distances for each simulation, given in Table 1; note that these distances are larger for simulations in which the adduct adopts the anti conformation compared with those with the adduct in the syn conformation and the control unmodified system. The resulting displacement of amino acids from their normal positions suggests that the incorporation of a nucleotide in the anti conformation paired with anti (ϩ)-ta-[BP]G should occur with diminished efficiency.
It is of interest to determine the effect of higher temperature on the outcome of these simulations in light of the temperature dependence of the experimentally determined k cat values. Each system simulated at 55°C is relatively stable and the stability of the simulations is illustrated by root mean square deviation analyses, given in supplemental Fig. S1. In the control simulation with unmodified DNA, the solute has an average heavy atom root mean square deviation of 2.2 Ϯ 0.2 Å from the starting structure. The nascent base pair remains undistorted, the two bases are coplanar and stacked with the ϩ1 base pair (see Fig. 3, right-hand panel), and they remain stably hydrogen bonded throughout the simulation. Supplemental Fig. S4 shows the hydrogen bonding distance and angle of the hydrogen bonds between the nascent base pair in each simulation. The stability of the unmodified system at 55°C, including the nascent base pair and its intact hydrogen bonds, is similar to that at 37°C (28) and demonstrates that the undamaged Dpo4 ternary complex is not distorted by the higher simulation temperature.
In contrast to the unmodified control, the nascent base pairs in the 55°C simulations with (ϩ)-ta-[BP]G in the anti conformation do not fare as well as their counterparts at 37°C, as shown in Fig. 3a. Whereas all three hydrogen bonds are intact in the case of the nascent base pair in the anti (ϩ)-ta-[BP]G: dCTP simulation at 37°C, two of these hydrogen bonds are disrupted with the base pair sheared at 55°C, as shown in Figs. 4a and 5a. The hydrogen bonding distances and angles are shown in supplemental Fig. S4a. In contrast to the analogous simulation at 37°C, the Watson-Crick alignment of the incoming dCTP at 55°C is imperfect; only one hydrogen bond is intact, and stacking of the nascent base pair with the neighboring pair is distorted (Fig. 3a). However, this anti (ϩ)-ta-[BP]G opposite dCTP remains a better accommodation than the syn (ϩ)-ta-[BP]G opposite dCTP at 55°C (see below).
The other three incoming anti dNTPs do not participate in hydrogen bonding interactions to any significant extent with the template anti (ϩ)-ta-[BP]G at 55°C; in contrast, at 37°C, both dATP and dTTP form stable hydrogen bonds with the anti-modified guanine during the entire simulation (28). At the higher temperature, two hydrogen bonds between dATP and anti (ϩ)-ta-[BP]G are in principle possible, but are not formed during the simulation of the anti purine-purine pair. However, one transient hydrogen bond is formed between anti (ϩ)-ta-[BP]G and dGTP as well as with dTTP, but only for 17 and 13% of the simulations, respectively (Fig. 5a). Furthermore, at 55°C all three nascent anti (ϩ)-ta-[BP]G:dATP, dGTP, and dTTP base pairs are distorted (Figs. 3a and 4a) and the incoming dNTP is not stacked with the 3Ј-terminal base of the primer. In the case of the anti (ϩ)-ta-[BP]G:dGTP nascent base pair, a highly distorted hydrogen bond forms between dGTP N 2 and (ϩ)-ta-[BP]G N 3 (Figs. 3a and 4a). The two base moieties of the nascent base pair are not aligned in the same plane and assume a staggered orientation relative to one another in each of the anti (ϩ)-ta-[BP]G:dATP, dGTP, and dTTP simulations (Fig.  3a). The incoming nucleotide is positioned on the 3Ј side of the damaged base in the anti (ϩ)-ta-[BP]G:dATP simulation, and on its 5Ј side in the anti (ϩ)-ta-[BP]G:dGTP and dTTP simulations at 55°C, as shown in Fig. 3a. In contrast, the nascent base pairs remain co-planar and stacked with the neighboring bases during the analogous dATP and dTTP simulations at 37°C. The distortions in the relative orientations of the nascent base pairs at 55°C suggest that the incorporation of any of the mismatched nucleotides (dATP, dGTP, or dTTP) in the anti conformation, would be less efficient opposite anti (ϩ)-ta-[BP]G than opposite syn (ϩ)-ta-[BP]G (see below). syn Purine dNTPs-Whereas there is no significant hydrogen bonding in the case of the anti (ϩ)-ta-[BP]G:anti dATP or dGTP nascent base pairs, significant hydrogen bonds are observed when dATP or dGTP assume a syn-glycosidic bond conformation at 55°C (Figs. 4 and supplemental S4). However, the nascent base pairs are more distorted than at 37°C (Fig. 3b),  55°C (pink). Additional color code for 55°C simulations: template, yellow; dCTP, red; dATP, green; dGTP, orange; dTTP, brown. Alignments were carried out using all residues that came within 6 Å of the damaged base in the anti (ϩ)-ta-[BP]G:dCTP simulation; the same residues and alignment protocol were utilized for the alignment of each system. The alignment of the unmodified systems is also given for comparison. Hydrogen atoms are not shown for clarity. which is evident from the angles between the planes of the bases shown in Figs. 4b and supplemental S3 and disrupted stacking with the ϩ1 base pair, shown in Fig. 3b. In both the anti (ϩ)ta-[BP]G:syn dATP and dGTP simulations at 55°C, two hydro-gen bonds form between the incoming nucleotides and the modified guanine base, shown in Fig. 5b. However, the planes of the syn dNTPs are rotated so that they are no longer parallel to the planes of the guanine moieties and are no longer stacked with the base on the 3Ј end of the primer. The angle between the planes of the bases of the nascent base pair in the unmodified simulation is Ϫ17 Ϯ 9°, whereas that in the anti (ϩ)-ta-[BP]G:syn dATP and dGTP simulations are Ϫ85 Ϯ 26°and Ϫ44 Ϯ 10°, respectively. This change in relative base orientations observed in the 55°C simulations is not entirely due to changes in the glycosidic torsion angles of the incoming dNTPs, as shown in supplemental Fig. S2; rather it is a result of a combination of smaller concerted changes in the sugar-phosphate backbone torsion angles. In contrast, the nascent base pairs in the anti (ϩ)-ta-[BP]G:syn dATP/dGTP simulations remain co-planar and stacked with the neighboring bases during most of the simulations at 37°C (28).

The Mismatched Bases are Markedly Better Accommodated Opposite syn (؉)-ta-[BP]G than Opposite anti (؉)-ta-[BP]G at 55°C, but dCTP Is More Favorable Opposite anti (؉)-ta-[BP]G
At 55°C, simulations of the mismatched bases opposite the (ϩ)-ta-[BP]G adduct in the syn conformation, with the BP residue positioned in the major groove, had nascent base pairs that were significantly more co-planar and stacked with the ϩ1 base pair, compared with their anti (ϩ)-ta-[BP]G counterparts, as shown in Fig. 3c. Furthermore, hydrogen bonding between the damaged base and incoming mismatched dNTPs was more stable with (ϩ)-ta-[BP]G in the syn conformation. An ion-mediated electrostatic interaction, depicted in Fig. 5(c), was also found between dTTP O-4 and syn (ϩ)-ta-[BP]G O-6 at 55°C, which further stabilizes this base pair; the distance and angles of this interaction are detailed in supplemental Fig. S5. Distances between P ␣ and O-3Ј often adopt near reaction ready ranges (ϳ3.1-3.6 Å)  at 55°C with syn (ϩ)-ta-[BP]G in the case of dATP and dGTP, occasionally for dTTP, but rarely for dCTP, as shown in Fig. 6.
Overall, the syn (ϩ)-ta-[BP]G:mismatch systems are structurally stabilized relative to their counterparts with anti (ϩ)-ta-[BP]G. The mismatched dNTPs are all distorted opposite anti (ϩ)-ta-[BP]G, and significant distortion of the nascent base pair even occurs when the purine incoming dNTPs are modeled into the syn conformation opposite anti (ϩ)-ta-[BP]G. Therefore, at 55°C, our results suggest that mismatched bases are incorporated significantly more opposite syn (ϩ)-ta-[BP]G than opposite anti (ϩ)-ta-[BP]G. In contrast, dCTP is better accommodated opposite anti (ϩ)-ta-[BP]G even at 55°C due to retention of close to the Watson-Crick alignment, with one hydrogen bond intact. Table 2 shows that the incoming dCTP in the syn (ϩ)-ta-[BP]G:dCTP simulation has an increased solvent-exposed surface area compared with that of the other syn (ϩ)-ta-[BP]G simulations despite the smaller size of this base compared with the purines; this reflects the distortion observed in this system at 55°C involving a shift of the dCTP toward the minor groove of the active site, as shown in Fig. 4c. The syn guanine is pyrimidine-like in the active site (63), producing poorer accommodation of pyrimidines than purines opposite the lesion; for dTTP as noted above, a Na ϩ -mediated electrostatic interaction aids in providing stability. The better structural organization of anti (ϩ)-ta-[BP]G:dCTP suggests that dC is better incorporated opposite anti (ϩ)-ta-[BP]G than opposite syn (ϩ)-ta-[BP]G. This is particularly supported by the frequent sampling of the near reaction ready P ␣ to O-3Ј distance (ϳ3.1-3.6 Å) in the anti, but not the syn (ϩ)-ta-[BP]G:dCTP simulations (Fig. 6).

Relationships between Experimental k cat Values and Molecular Modeling
Studies-Comparisons of experimental data with the results of molecular modeling studies are of great potential interest because the latter may lead to predictions that can be tested experimentally, thus allowing for a deeper understanding of the nucleotidyl transfer reaction. In the molecular modeling studies, we have used molecular dynamics simulations to visualize structures at the active site of the polymerase in ternary dNTP⅐primer-template⅐polymerase complexes. The implicit assumptions are that successful dNTP incorporation is   Temperature Dependence of dNTP Opposite a DNA Adduct most likely for conformations of the interacting partners and critical amino acids that are most similar to those of normal dNTP insertion opposite an unmodified guanine template residue. The experimentally measured rate constants of dNTP incorporation are exemplified by steady-state measurements of k cat , a rate constant that reflects the slowest, rate-determining event in a series of mechanistic steps (74,75). The nature of the rate-determining step is not always easy to determine, as recently described for the bypass of the (ϩ)-ta-[BP]G and other bulky adducts by the A family polymerase T7 exo- (34). In the case of Dpo4, Fiala and Suo (76) found that the rate-limiting step for incorporation of the correct dNTP opposite a normal template base is a protein conformational change that precedes the phosphodiester bond formation chemical step. In contrast, the chemical step is rate-determining when the incorrect dNTP is incorporated opposite a normal DNA template base. In our experiments, the rate of incorporation of the dNTPs opposite the (ϩ)-ta-[BP]G lesion (the Ϫ1 to 0 step in Fig. 2a) is slow, the k cat values are of the order of 10 Ϫ3 min Ϫ1 (Fig. 2c), and there is no burst phase (i.e. product formation increases linearly with time under conditions of conversion Ͻ20%; typical examples are shown in supplemental Fig. S6). These are all hallmarks of a rate-determining chemical step or a rate-limiting conformational change that precedes phosphodiester bond formation (34). The results of the molecular modeling studies reported here and earlier (28) are relevant to experimental values of k cat if the chemistry step is rate-determining. However, it is also possible that a protein or nucleic acid conformational change that precedes the chemistry step and leads to the correct alignment of the reaction partners in the active site is rate-determining. Keeping these uncertainties and caveats in mind, we have examined possible relationships between the experimentally measured values of k cat and the structural insights from our molecular modeling studies. Our reasoning is that active site distortions produce impeded polymerase function, preventing correct assembly of the active site components.
Effect of Temperature on the anti 7 syn Equilibrium and Translesion Bypass-Increasing the reaction temperature from 37 (28) to 55°C increases the k cat for insertion of all four nucleotides opposite (ϩ)-ta-[BP]G (Fig. 2c). The molecular dynamics simulations show that the presence of the carcinogen moiety in the minor groove in the anti (ϩ)-ta-[BP]G simulations at both temperatures causes the cleft between the little finger and fingers and palm domains to widen relative to the unmodified system, as determined from the average distance between two residues in the palm and little finger domain in the simulations (Table 1). However, the major groove/syn simulations scarcely show this distortion (supplemental Fig. S7). Translesion bypass of the (ϩ)-ta-[BP]G adduct in the syn glycosidic conformation is likely to be more facile because the BP rings are positioned on the more spacious major groove side of the nascent base pair. The higher rate of nucleotide insertion opposite (ϩ)-ta-[BP]G at 55°C as compared with 37°C can be explained by a shift of the anti 7 syn equilibrium toward the syn conformation, assuming this process is associated with an activation energy and is thus temperature-dependent. The syn conformation of (ϩ)-ta-[BP]G has previously been observed at a primer-tem-plate junction in high resolution NMR studies (77). Rotation of the glycosidic bond of (ϩ)-ta-[BP]G from the anti to the syn domain may occur when the damaged base is positioned in the single strand region of the template. The template strand threads into the active site via the major groove side of the primer-template (Fig. 3, right panel) and it is relatively unimpeded from rotation about its glycosidic torsion.
Raising the temperature from 37 to 55°C increases the k cat values for the insertion of mismatched dNTPs opposite (ϩ)-ta-[BP]G significantly more than the insertion of the correctly matched dCTP. The k cat for insertion of a complementary dCTP opposite an unmodified G and (ϩ)-ta-[BP]G are both increased by a factor of only ϳ2 as the temperature is increased from 37 to 55°C. 5 However, the k cat 55°C/37°C ratios for the insertion of the mismatched nucleotides dATP, dGTP, and dTTP are ϳ7-10 and are thus significantly higher. A greater preference for the syn conformation of (ϩ)-ta-[BP]G explains the preferred insertion of the mismatched nucleotides over the correct dCTP at 55°C; in the syn conformation of (ϩ)-ta-[BP]G, the insertion of dATP, dGTP, and dTTP opposite the damaged base is favored over the insertion of dCTP because the active site is less well organized for reaction in the syn (ϩ)-ta-[BP]G:dCTP system (Fig. 6b). dCTP is more likely to be inserted opposite anti (ϩ)-ta-[BP]G than syn (ϩ)-ta-[BP]G because the incoming dCTP is better accommodated in the active site at both temperatures (Figs. 5a and 6a), with all three Watson-Crick hydrogen bonds present at 37°C, and one remaining intact at 55°C. In contrast, dATP, dGTP, and dTTP, incapable of Watson-Crick base pairing with the damaged base, are better accommodated opposite syn compared with anti (ϩ)-ta-[BP]G at both temperatures, but more so at 55°C (Figs. 3 and 6). At 37°C, anti/syn dATP, syn dGTP, and anti dTTP are well accommodated opposite anti (ϩ)-ta-[BP]G, but all of the mismatched bases, including syn dATP and dGTP, become very distorted opposite anti (ϩ)-ta-[BP]G at 55°C (Fig. 3b). The near reaction ready P ␣ to O-3Ј distance (ϳ3.1-3.6 Å) is sampled much more frequently in the simulations with syn (ϩ)-ta-[BP]G opposite the mismatches, but with anti (ϩ)-ta-[BP]G opposite normal partner dCTP. Therefore, a shift of the equilibrium toward the syn conformation of (ϩ)-ta-[BP]G favors the insertion of the mismatched bases over the insertion of the correct dCTP. In contrast, at 37°C, all of the dNTPs are accommodated reasonably well opposite anti or syn (ϩ)-ta-[BP]G, with the exception of dGTP, which is poorly accommodated opposite anti (ϩ)-ta-[BP]G.
Conclusion-Our results show that the rate of mismatched nucleotide incorporation is greater than the rate of correct dC insertion at 55°C, whereas at 37°C there is little selectivity. Enhanced adoption of the syn conformation at the higher temperature can account for the increased propensity to accommodate mismatches. Our structure-function analyses also suggest that changes in reaction temperature may significantly alter enzyme specificity by increasing conformational flexibility and/or allowing access to conformations that are less favored at lower temperature.