Effect of the Y955C Mutation on Mitochondrial DNA Polymerase Nucleotide Incorporation Efficiency and Fidelity
The human mitochondrial DNA polymerase (pol \xce\xb3) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol \xce\xb3 results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson\xe2\x80\x99s disease. In single turnover kinetic studies, we show that the Y955C mutation resulted in a decrease in the maximum rate of polymerization and an increase in the Km for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 \xce\xbcM\xe2\x88\x921s\xe2\x88\x921, respectively, representing reductions of 30-, 110- and 1300-fold relative to wild-type enzyme. The fidelity of incorporation was reduced 6- to 130-fold; largely due the significant decrease in the specificity constant for correct dATP:T incorporation. For example, kcat/Km for forming a TTP:T mismatch was decreased tenfold from 0.0002 to 0.00002 \xce\xbcM\xe2\x88\x921s\xe2\x88\x921 by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to wild-type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role of Y955 in stabilizing A:T base pairs at the active site of pol \xce\xb3 and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of dATP incorporation opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.
<!>Cloning and Plasmid Construction for the Large Subunit of Polymerase \xce\xb3 (p140)<!>Purification of Small Subunit (p55) and Polymerase \xce\xb3 Y955C mutants<!>Preparation of DNA<!>Single Turnover Experiments<!>Global Fitting<!>A note on nomenclature<!>Free energy of discrimination<!>Calculation of kcat and Km<!>Kinetics of single nucleotide incorporation measured by quench-flow experiments<!>Kinetics of misincorporation<!>Incorporation of 8-oxo-dGTP<!>Global Fitting/Contour analysis<!>Discussion<!>Physiological effects
<p>Understanding the molecular basis for disease has become an increasingly important part of biochemical studies as genetic testing becomes more common. Identification of the proteins or genes responsible for a particular disease could allow for better treatments and/or earlier intervention. Deletions or mutations in human mitochondrial DNA polymerase (pol γ) have been correlated with various mitochondrial disorders, including mtDNA depletion syndrome, Alpers Syndrome, and progressive external opthamalplegia (PEO) (1). Symptoms of Alpers Syndrome include liver disease and refractory seizures, while patients with PEO present with progressive weakness of the external ocular muscles and skeletal myopathy (2, 3).</p><p>Mitochondrial DNA (mtDNA) replication is performed by a replisome comprised of a nuclearly-encoded catalytic subunit, two subunits of the processivity factor (p55), the single-stranded DNA binding protein, and a DNA helicase (4). The crystal structure of the holoenzyme, a heterotrimer comprised of the catalytic subunit of the polymerase and a dimer of p55, was recently solved (5). Like other Family A polymerases, the mtDNA polymerase structure resembles a right hand comprised of thumb, fingers, and palm domains. Recent studies on two other A family polymerases, HIV reverse transcriptase and T7 DNA polymerase, have guided our work on pol γ, providing insight into the mechanism of catalysis as well as the potential mechanisms of discrimination against incorrect nucleotides and the role of nucleotide-induced conformational changes in selectivity (6, 7). The pathway of nucleotide incorporation can be simplified to that shown in Scheme 1, where EDn represents a complex of enzyme with DNA that is n bases long, and N represent a nucleoside triphosphate.</p><p>In this simplified scheme, the ground-state binding is represented by the term 1/K1 (Kd), while the rate of chemistry is described by kpol. Normally pyrophosphate release and translocation to allow the binding of the next nucleotide are much faster than incorporation (8–10). Accordingly, the rate of incorporation measured in a single turnover experiment defines the kinetics governing sequential nucleotide incorporation events during processive synthesis. Accordingly, the values of kpol/Kd measured in single turnover kinetic studies define kcat/Km for processive synthesis. Moreover, the apparent Kd measured in these studies should be more generally described as a Km value, which approaches a true Kd when the chemistry step is slow (6, 7). Throughout the text, we will refer to the results from our single turnover kinetic studies in terms of kcat and Km values to eliminate the misconception that the results define Kd.</p><p>The crystal structure of the ternary complex of pol γ has yet to be solved, and so comparisons between pol γ and T7 DNA polymerase have been helpful in identifying the potential roles of particular residues. As summarized in Fig. 1, catalytic residues are conserved in comparing T7 DNA polymerase and pol γ thereby allowing homology modeling based upon the T7 DNA polymerase ternary E-DNA-nucleotide complex (11, 12) to suggest possible interactions at the active site. Within the active site of pol γ, homology suggests that residue R943 contacts the γ phosphate, while Y951 and H932 each contact the β-phosphate, leaving K947 to make contact with the α-phosphate, as confirmed by recent mutagenesis studies (13). Tyrosine 955 is not involved directly in contacts with the phosphate backbone; rather, it appears that it participates in a hydrogen-bonding network involving a glutamate residue in the palm of the enzyme (E895 in pol γ, not shown). This hydrogen-bond network may be essential not only for maintaining the architecture of the polymerase active site but also for the recognition of the correct incoming nucleotide. Mutation of Y530 to a phenylalanine in T7 DNAP resulted in a loss of discrimination through a lower affinity for the incoming nucleotide rather than a decrease in the rate of polymerization (14).</p><p>Autosomal dominant forms of PEO usually involve mutations in pol γ that are in the active site of the polymerase. Patients with the Y955C mutation present with early onset autosomal dominant PEO in their thirties (15). These patients often later present with Parkinson's disease and in women, premature ovarian failure (15–17). MtDNA in skeletal muscle cells of these patients shows some deletions, though a large proportion of the mtDNA is intact, indicating some level of complete replication by the polymerase (17). In yeast, a mutant of Mip1 homologous to the human polymerase gamma mutant Y955C leads to increased petite frequency, suggesting mitochondria malfunction (18). In addition, qualitative studies have shown that both the mutation and deletion frequency are increased with this mutant (3, 19).</p><p>Oxidatively damaged guanosine, in the form of 8-oxo-deoxyguanosine, accumulates in the mitochondria and contributes to the formation of mutations (20). The 8-oxo-dG can base pair with either a cytosine in the anti-conformation or with an adenosine in the syn-conformation, leading to frequent mutations (21). Pol γ uses a novel mechanism to reduce the rate of incorporation of 8-oxo-dGTP by slowing the rate of pyrophosphate release allowing the chemical reaction to come to equilibrium at the enzyme active site (21). Therefore, it is important to establish which residues are responsible for this slow pyrophosphate release, as they affect discrimination against 8-oxo-dGTP. Therefore we have examined the kinetics of incorporation by the Y955C mutant as part of this study. Moreover, if the Y955C mutant incorporated 8-oxo-dGTP at a higher rate, that could account for the observed physiological effects of this mutation.</p><p>Understanding the molecular basis for diseases linked to mtDNA replication requires accurate kinetic analysis to quantify the effects of the mutation. Although many mutations in pol γ have been characterized previously (12, 22, 23), we show in this study that the published results are unreliable. Our new results provide a basis to correlate the biochemistry of mtDNA replication with physiology.</p><!><p>The creation of the Y955C mutant on an exonuclease deficient background (D198A/E200A) on the pUC19 plasmid (New England Biolabs) was performed using two-step PCR using primers flanking SacII and NotI restriction sites. The mutagenic primers used were the following: 5′ CAA TTA CGG TCG TAT TTG CGC GCA GGT CAG CCG 3′, complementary strand: 5′ GCT GAC CTG CGC CGC AAA TAC GAC CGT AAT TGA AAA T 3′. The SacII and NotI restriction sites were used to insert the PCR-generated fragment into the gene. A NotI/BglII digest was then used to remove the gene from pUC19.1 for ligation into the pBacPak9 vector (Clontech). Creation of baculovirus containing the gene for Y955C pol γ was as previously described (13).</p><!><p>The small subunit (p55) was expressed using the pET43.1a vector with a histidine-tag in E. coli Rosetta 2 (DE3) and then purified as previously described (13). For purification of the large subunit of the polymerase with the Y955C mutation, the cells were lysed by re-suspension in a buffer containing 0.32 M Sucrose, 10 mM HEPES (pH 7.5), 3 mM CaCl2, 2 mM MgAc•4H20, 0.1 mM EDTA. Protease Inhibitor Cocktail IV (EDTA-free, AG Scientific) was added to the lysis buffer. The lysate was clarified by centrifugation at 1,500 × g for 15 minutes. DNA was removed by increasing the salt to a final [KCl] of 0.5 M. The lysate was again centrifuged in a Beckman Coulter Ultracentrifuge at 31,000 × g in a Ti45 rotor. Next, the lysate was subjected to a Ni-NTA column (QIAgen) via the batch method. The protein was bound to the resin pre-equilibrated with 20 mM HEPES (pH 7.5), 5 mM imidazole, 200 mM KCl, and 5% glycerol by stirring on ice. The beads were collected via centrifugation at 1500 × g for 10 minutes, and then washed with 20 mM HEPES (pH 7.5), 200 mM KCl, 20 mM imidazole, 5% glycerol. The beads were then poured into a column and the protein was eluted with 20 mM HEPES (pH 7.5), 100 mM KCl, 200 mM imidazole, 5% glycerol. The fractions containing the polymerase were pooled and diluted before loading onto an SP Sepharose column (Amersham Biosciences) with a flow rate of 1ml/min in buffer A containing 20 mM HEPES (pH 7.5), 30 mM KCl, 1 mM DTT, 1 mM EDTA, 5% glycerol. Buffer B contained 20 mM HEPES (pH 7.5), 700 mM KCl, 1mM DTT, 1 mM EDTA, 5% glycerol. The protein was eluted with a step-gradient from 0–100% B. The fractions containing polymerase gamma were pooled, concentrated via a centrifugal concentrator with a 50 kDa cutoff (GE Healthcare), and dialyzed against the final storage buffer containing 50 mM Tris-HCl (pH 8.4), 100 mM NaCl, 1 mM DTT, 50% glycerol. Concentrations of active enzyme were obtained by active site titration of the burst amplitude versus DNA concentration.</p><!><p>Oligonucleotides were purchased from IDT DNA Technologies and purified using 15% acrylamide/7M Urea denaturing PAGE gels. A 25-mer primer and a 45-mer template (Template 1 shown in Table I) were adopted from previous studies for misincorporation assays (13). Templates 2 and 3 were used for incorporation assays involving 8-oxo-dGTP. The 25-mer was 5′ 32-P labeled with T4 polynucleotide kinase as recommended by the manufacturer (New England Biolabs). Excess γ-32-P –ATP was removed by a Bio-Spin 6 gel filtration column (BioRad), and the amount of labeled DNA was quantified by TLC. The labeled 25-mer and unlabeled 45-mer were mixed in a 1:1 molar ratio and annealed by heating to 95 degrees for 5 minutes, followed by slow cooling to room temperature.</p><!><p>All experiments were performed at 37° C in a solution containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 12.5 mM MgCl2.. To eliminate complications of multiple turnovers, reactions were performed under single turnover conditions, with enzyme in excess and limiting DNA substrate. For fast reactions, assays were performed with a KinTek RQF-3 Rapid Quench Flow apparatus. For both correct incorporation and misincorporation assays, the holoenzyme (200 nM to 300 nM large subunit and 0.8 to 1.2 μM small subunit) were pre-incubated with 25/45-mer DNA (140 nM-200 nM) in reaction buffer without magnesium chloride and loaded into one syringe. The other syringe contained the nucleotide (0.5 μM to 500 μM for correct incorporation, and 25 μM to 5 mM for incorrect incorporation) in reaction buffer with magnesium chloride. After rapid mixing, the reaction was quenched with 0.5 M EDTA. Misincorporation assays requiring longer time points were performed by manual mixing. Reactions involving 8-oxo-dGTP were performed with either rapid quenching or hand quenching with concentrations varying from 1 μM to 1 mM 8-oxo-dGTP.</p><p>The products were resolved on 15% acrylamide/7M denaturing PAGE gels. The gels were dried and exposed to a PhosphorImager screen. The screen was then scanned by a Typhoon Scanner (GE Healthcare Life Sciences) to reveal product bands. The amount of product (26-mer) was quantified versus the amount of starting material to determine fraction of product.</p><!><p>All data were fit globally based upon numerical integration of rate equations using the KinTek Explorer program (KinTek Corporation) (24, 25). Nonlinear regression was used to find optimal parameters in fitting each set of data to a single model. While fitting data, previously estimated rates of DNA binding and release were used in constructing a comprehensive model including all known steps. For example, Scheme 1 was expanded to: 
E+Dn⇄koffkonEDnEDn+N⇄k−1k1EDnN⇄k2EDn+1+PPi</p><p>The DNA binding and dissociation rates for both substrate and product DNA (Dn, Dn+1) were set to be kon = 20 μM−1s−1 and koff = 0.02 s−1 to satisfy the steady state DNA release rate and the Kd,DNA of 10 nM (26). The rate of nucleotide binding is thought to be limited by diffusion, so k1 was set to 100 μM −1s−1. By allowing the dissociation rate to vary (k−1), the equilibrium constant was calculated, K1 = k1/k−1. Thus, to fit a full concentration series, only k−1 and k2 were adjust to fit the family of curves and thereby define the apparent Kd (Km) and kpol (kcat), respectively. To account for slight variations in the data, enzyme or DNA concentration were adjusted slightly (±10%) to derive best fits. Standard error estimates were obtained by nonlinear regression and were checked using confidence contour analysis. The upper and lower limits were derived based on a chi2 threshold of 20%, as previously described (25). In all cases, the error estimates derived by nonlinear regression were similar to the estimates based upon the confidence contour analysis, verifying that the parameters were well constrained.</p><p>In presenting the results in each of the figures, data at long times were omitted from the graph in order to illustrate more clearly the data at shorter times. Although not displayed, data at longer times were included in the data fitting. In particular, the longer time points help to define the amplitude of the reactions at the lower concentrations as part of the global fitting.</p><!><p>We reserve the term, "rate constant" to only refer to an "intrinsic rate constant" for an individual step in a reaction pathway (i.e., that represented by a lower case k over an arrow). Moreover, we see little utility in the distinction between a rate derived by an initial velocity (slope as the change in product with time) and a rate measured by fitting to an exponential function and do not adhere to the standard that the latter should be referred to as a "rate constant." Although clearly having distinct meanings and units, both are methods to measure the rate of a reaction. Moreover, it is often misleading to presume that an Eigenvalue derived by fitting data to an exponential function is an intrinsic rate constant. In the process of fitting data, we derive rates of reaction in units of s−1, and because these measurements do not necessarily resolve individual rate constants, we refer to them as rate measurements.</p><!><p>Discrimination against each mismatch was calculated from: 
D(kcat/Km)correct(kcat/Km)mismatch which is equal to the reciprocal of the frequency of misincorporation (fidelity). We computed the apparent change in free energy due to the Y955C mutation reflected in the changes in kcat and Km according to the following equations: 
ΔΔGc=−RTln(kcatY955Ckcatwt)
ΔΔGm=−RTln(KmwtKmY955C)</p><p>The net change in the specificity constant is the sum of the contributions from kcat and Km on the free energy scale.</p><!><p>After fitting data in Fig. 5C to Scheme 2, we computed kcat and Km from the following: 
kcat=k2k3k2+k−2+k3Km=k2k3+k−1(k−2+k3)k1(k2+k−2+k3)kcat/Km=k1k2k3k2k3+k−1(k−2+k3)</p><!><p>Single-nucleotide incorporation experiments were performed to determine the parameters for correct nucleotide incorporation by the Y955C mutant. The experiments were performed under single-turnover conditions in that the concentration of enzyme exceeded the concentration of DNA. The nucleotide concentration dependence of the rate was analyzed to provide estimates of an apparent dissociation constant (Kd,app = Km) and a maximum rate of nucleotide incorporation, (kpol = kcat) according to Scheme 1. Fig. 2 shows the time dependence of product formation at several nucleotide concentrations to define the kinetics of incorporation of dATP:T, dGTP:dC and TTP:dA. To avoid confusion, we use the notation dATP:T to represent the incorporation of dATP opposite a template T, for example. The data in the experiment were globally fit to the mechanism shown in Scheme 1 based upon numerical integration of the rate equations to obtain values for the apparent nucleotide dissociation constant and the maximum rate of incorporation (24).</p><p>Several underlying assumptions govern the fitting of the data to the model in Scheme 1. Ground state nucleotide binding is thought to occur as a rapid equilibrium, which was modeled with the initial collision of the nucleotide and enzyme occurring at a rate limited by diffusion (100 μM −1s−1) in order to define an apparent dissociation constant, Kd,app (Km). Second, the rate-limiting step is believed to be polymerization, while conformational changes preceding chemistry, pyrophosphate release and translocation are thought to be fast, based upon more detailed studies on homologous enzymes (6, 7). Accordingly, the ratio of kpol/Kd,app defines kcat/Km, the specificity constant governing sequential nucleotide incorporation events during processive synthesis.</p><p>The kinetic parameters for correct incorporation are summarized in Table 2 and compared to the values for wild-type enzyme (27). The Y955C mutation affected not only the rate of polymerization, dropping as much as a factor of 20, but kcat/Km was greatly affected. The effect on kcat/Km was most severe with dATP:T base pairs, mostly due to much weaker nucleotide binding. As summarized in Table 2, the Y955C mutation caused a 1300-fold reduction in kcat/Km for incorporation of dATP:T, while the effects on TTP:dA and dGTP:dC were only 110-and 30-fold, respectively.</p><!><p>The kinetics of misincorporation were measured to investigate the discrimination against mismatches by the Y955C mutant compared to the wild-type enzyme. In particular, we examined the incorporation of dCTP, TTP, and dGTP opposite a templating T. In many cases, multiple nucleotides were incorporated onto the primer strand such that products up to 32 nucleotides long were observed (see Fig. 3). When quantifying the product, all of the extended primers were included in the total concentration of product formed in order to define the rate of formation of the first mismatch. The concentration of product was plotted as a function of time (Fig. 4), and then fit globally to Scheme 1 to get the kinetic parameters listed in Table 3. The kinetic parameters for misincorporation for the wild-type enzyme were determined previously (28) and are listed for comparison. As shown in Table 3, there is a significant decrease in fidelity, 6-, 26, 120- and 50-fold for dGTP:T, dCTP:T, TTP:T and dGTP:dA mismatches, respectively.</p><!><p>The kinetics of incorporation of 8-oxo-dGTP were measured under single turnover conditions opposite a dCMP or dAMP templating base. The formation of product over time is shown in Figs. 5A and 5B, and fit to Scheme 1 to get the results summarized in Table 4. These data show no significant change in kcat/Km for incorporation of 8-oxo-dGTP opposite a template dC, but a 500-fold reduction for incorporation opposite a template dA, compared to wild-type.</p><p>It was important to evaluate whether the kinetics of incorporation of 8-oxo-dGTP followed the more complex pattern seen with the wild-type enzyme. That is, with the wild-type enzyme the kinetics show a clear nucleotide concentration dependence of amplitude as shown in Fig. 5C, reproduced from (21). Incorporation of 8-oxo-dGTP by the wild-type enzyme follows Scheme 2, with a slow pyrophosphate release leading to accumulation of the product complex (EDn+1PPi) in equilibrium with bound substrate. This leads to a distinct kinetic signature where the amplitude of the pre-steady state burst is dependent upon nucleotide concentration.</p><p>In Fig. 5C, we reproduce the data from the Hanes & Johnson 2006 paper, but now fit the data globally to the model shown in Scheme 2. Confidence contour analysis (Fig. 6B) justifies the fitting of the data to four rate constants by showing that all four rate constants are well constrained.</p><p>Incorporation of 8-oxo-dGTP opposite a templating dC by the Y955C mutant (Fig. 5B) does not show the same pattern as seen for the wild-type enzyme. That is, the curves for the Y955C mutant share the same end-point derived by fitting the data globally, and the data can be accounted for sufficiently by the model shown in Scheme 1. Thus, unlike the wild-type enzyme (Fig. 5C), the concentration dependence of incorporation of 8-oxo-dGTP by the mutant gave no evidence to suggest that the reaction was followed by slow pyrophosphate release. The rate of misincorporation of a 8-oxo-dGTP:dA base pair by the Y955C mutant was 500-fold slower (Table 4, kcat/Km = 0.0004 μM−1s−1) than by the wild-type (kcat/Km = 0.2 μM−1s−1); and thus, the mutant showed increased discrimination against misincorporation of 8-oxo-dGTP. The incorporation of 8-oxo-dGTP:dA was comparable to the rate of misincorporation of dGTP:dA (Table 3, kcat/Km = 0.0003 μM−1s−1). Overall, the data suggest that the physiological effects of the Y955C mutation are not due to increased rates of incorporation of 8-oxo-dGTP.</p><!><p>Each set of curves was globally fit using the KinTek Explorer (24, 25). The global fitting routine is based on numerical integration of rate equations, allowing the parameters to be derived by directly fitting to a complete model. In this type of fitting, both the rate and the amplitude of the reaction are taken into account, eliminating the simplifications of equations and errors that are common during traditional fitting. This has proved useful in fitting data where there is a clear nucleotide concentration dependence of the amplitude, or data where the nucleotide concentration is comparable to that of enzyme. It is necessary to ensure that the model is not overly complex, and that the data are able to support the definition of each step in the model. We address this by performing confidence contour analysis, whereby the constants are systematically varied and we estimate the extent to which each parameter in a model is constrained by the data.</p><p>In contour analysis (Fig. 6A), the red zone represents a range of values that provide a good fit to the data given in Fig. 5A. The margin shown by the yellow band between the red and green zones defines a boundary that represents a 20% increase in chi2, which is used to estimate the upper and lower confidence intervals on each of the parameters (25). In the case of 8-oxo-dGTP incorporation by the Y955C mutant (Fig. 5A), we can see that the parameters are well constrained by the data to support the mechanism shown in Scheme 1. The upper and lower confidence limits have been compared to the standard error estimates derived by nonlinear regression. Because the two methods of estimating errors agreed for the data reported here, we omitted the upper and lower limits from the Tables for clarity. Note also that the ratio of kcat/Km is known with greater certainty than either parameter, kcat or Km, as revealed by the linear relationship between k2 and k−1 on the confidence contour. This is equivalent to stating that the slope of a rate versus concentration curve is known with greater certainty than the kcat/Km value computed from the ratio of the kcat and Km values.</p><p>In Fig. 6B, we show the confidence contours derived from global fitting of the data in Fig. 5C. This analysis demonstrates that all four rate constants were well constrained in fitting the data to Scheme 2 based upon simultaneously fitting the rate and amplitude dependence of the observed reaction. As summarized in the legend to Fig. 5, the rate constants derived by global fitting are comparable to those obtained by conventional analysis, but the error limits are better defined by the global fitting and the concentration dependence of the rate and amplitude are fit simultaneously, rather than separately.</p><!><p>In all Family A polymerases, there is a tyrosine residue at the end of the helix containing several catalytic residues. Studies involving the T7 DNA polymerase have suggested that this residue plays a role in stabilizing the incoming nucleotide, with the phenyl ring of the tyrosine stacking with the nucleotide, and the hydroxyl group forming hydrogen bonds that help to stabilize the architecture of the active site (11). Mutation of the homologous residue (Y530) to phenylalanine in T7 DNA polymerase resulted in an increase in the dissociation constant for incoming nucleotides with only a modest effect on the rate of polymerization (14) and a corresponding decrease in fidelity. As shown here, the mutation of tyrosine to a cysteine has a much more drastic effect. In particular, we observe a 30- to 1300-fold reduction in kcat/Km governing correct base pair incorporation due to the Y955C mutation. In addition, there was a 6 to 120-fold reduction in fidelity. In contrast, there was little change in the kinetics of incorporation of 8-oxo-dGTP opposite a template dC, but a 500-fold improvement in discrimination against a 8-oxo-dGTP:dA mismatch.</p><p>Previous studies of the Y955C mutation based upon steady-state methods in the Copeland laboratory have provided inconsistent results (12, 29). For example, steady state measurements gave values of kcat = 0.05 s−1, Km = 40 nM, and kcat/Km = 1.2 μM−1s−1 for incorporation of dGTP:dC by wild-type enzyme (30). In contrast, our methods provided values of kcat = 37 s−1, Km = 0.8 μM, and kcat/Km = 46 μM−1s−1 (Table 2). Thus, the specificity constant derived by steady state methods was 38-fold lower than what we observed in single turnover experiments. Comparing values for wild-type enzyme and the Y955C mutant reveals that the kcat/Km values derived by steady state methods in the Copeland laboratory were lower by factors ranging from 27- to 62,000-fold (30) compared to the results presented here. Most noteworthy, Copeland and his coworkers underestimated the rate of incorporation of 8-oxo-dGTP:dA by wild-type enzyme by a factor of 8,000-fold and thus they failed to properly assess the importance of mismatch formation with this oxidatively damaged nucleotide. In addition, they underestimated the specificity constant for incorporation of dGTP:dC by the Y955C mutation by 62,000-fold and accordingly overstated the magnitude of the defect. Similar discrepancies exist in the specificity constants for incorporation of nucleoside analogs used to treat HIV infections in comparing the results from pre-steady state (31) and steady state kinetics (32). In comparing the published results from two papers from the Copeland lab (12, 29), kcat values for wild-type enzyme were reported to be either 0.36 min−1 or 1.3 s−1. This 220-fold variation in kcat values reported for the wild-type enzyme engenders little confidence in the quality of data.</p><p>For DNA polymerases, steady state kcat and Km values are reduced by a factor approaching the processivity of the enzyme, which for pol γ is approximately 2,000 (26). Although, in theory, steady state measurements on a DNA polymerases should still provide a valid specificity constant (33) and may be adequate for enzymes with low processivity (34), in practice, experiments on pol γ would need to be designed to accurately measure a steady state Km value of ~0.5 nM to accurately assess correct incorporation kinetics. As a counterargument, one might propose that the single turnover kinetics overestimate the rates because the first turnover might be faster than subsequent turnovers. However, experiments to measure the kinetics of sequential nucleotide incorporation on several DNA polymerases have shown that the rate of incorporation of each nucleotide is comparable to that measured in single turnover experiments (9, 27, 35). Moreover, there is no evidence for a kinetically significant step between sequential nucleotide incorporation events, implying that pyrophosphate release and translocation are normally much faster than incorporation. Direct measurements on pol γ demonstrated that pyrophosphate release was kinetically correlated with incorporation, implying fast pyrophosphate release after chemistry (36, 37). Thus, the rate constants derived from single turnover kinetic studies accurately define the parameters governing sequential nucleotide incorporation events during processive synthesis and avoid the complications introduced in steady state measurements of a processive DNA polymerase. Moreover, pre-steady state experiments provide important quality control for the concentration of active enzyme in a preparation from the measured amplitude of the reaction. In surveying the literature on pol γ, it is our opinion that the results of steady state kinetics, in general, and those from the Copeland lab in particular, are unreliable and conclusions based upon their quantitative results must be reevaluated.</p><p>Family A polymerases typically have a two-step binding mechanism, where a conformational change is a critical component of nucleotide recognition (6, 7, 38). Studies on T7 DNA polymerase and HIV RT have shown that this conformational change is fast and establishes the specificity constant for correct incorporation. However, when the chemistry step is slow, the conformational change comes to equilibrium before incorporation (6, 7). Without any method to measure the conformational change in pol γ, it is difficult to interpret whether the apparent Kd represents a true dissociation constant or simply reflects the ratio of kcat and the net substrate binding rate. The issue is whether the binding steps come to equilibrium before the chemistry step. In either case, the apparent Kd provides an accurate estimate of the Km governing single nucleotide incorporation during processive synthesis and kpol/Kd defines the specificity constant, kcat/Km (6, 7). In studies on T7 DNA polymerase and HIV RT, it was shown that when the rate of chemistry is reduced by mutation or when incorporating a mismatch or a nucleoside analog, the binding steps come to equilibrium (6, 7). Accordingly, the slow rate of incorporation suggests that binding comes to equilibrium with the Y955C mutation, implying a significant affect of the Y955C mutation on nucleotide binding (Fig. 2 and Table 2).</p><p>The Y955C mutation resulted in reduced rate of polymerization and a decrease in the kcat/Km for correct incorporation. The effect of the mutation on the maximum rates of polymerization varied from 1.5- to 20-fold for correct incorporation (Table 2). However, the catalytic efficiencies (kcat/Km) were reduced 30 to 1300-fold. Thus, the effect of the Y955C mutation on the chemistry step was less significant and not correlated with the largest changes in kcat/Km. In particular, the specificity constant for incorporation of dATP:T was reduced 1300-fold with only an eightfold reduction in kcat. Thus, unlike wild-type enzyme, the specificity constant varied considerably for different base pairs. Perhaps the hydrogen bond network involving this tyrosine in the wild-type enzyme compensates for fewer hydrogen bonds in forming an A:T base pair compared to a G:C base pair, as suggested in early work on T7 DNA polymerase (14). Perhaps the stacking interaction between the tyrosine and the incoming base acts as an equalizer, allowing the rates of binding and polymerization to be less dependent on the type and size of the base. Cysteine is unable to compensate. Because of the positioning of Y955 (on the O-helix in T7 DNA polymerase), the interaction with the E895 residue may be critical in the ground state binding of the incoming nucleotide. The hydroxyl group of the tyrosine may stabilize the carboxyl group of the glutamate in the active site. In the T7 DNA polymerase, this glutamate selectively stabilizes the incoming TTP of a T:A base pair (14). This interaction may be reflected in Y955C pol γ as an increased Km for TTP or dATP and reduced rates of catalysis.</p><p>Another significant detrimental effect of the Y955C mutation is reduced fidelity. The biggest drop in fidelity occurred between a T:T mismatch from one error in 260,000 for wild-type compared to one in 2,200 for the Y955C mutant. This 120-fold decrease in fidelity may contribute to the physiological consequences of this mutation. While replicating DNA, the Y955C mutant slows when reaching a T in the template and one time in 2,200 it will incorporate a T:T mismatch. Perhaps equally important, we show that the Y955C mutant catalyzes multiple misincorporation events, unlike the wild-type enzyme (27).</p><p>We have also studied the kinetics of incorporating 8-oxo-dGTP opposite dC and dA in the template. Our single-turnover experiments suggest not only a low discrimination against 8-oxo-deoxygyanosine, but that the level of discrimination is greater against a 8-oxo-dGTP:dA misincorporation (Table 4) for Y955C compared to wild-type enzyme. The wild-type enzyme has an unusual mechanism to minimize incorporation of 8-oxo-dGTP opposite a dCMP base, creating an equilibrium by slowing the release of pyrophosphate and allowing time for the reverse reaction to compete with product release (21). This resulted in a nucleotide dependence of amplitude and ultimately slowing the kcat (Fig. 5C.). However, with the Y955C mutation, there was no evidence for the slow pyrophosphate release, but discrimination is lowered due to weaker nucleotide binding.</p><!><p>The Y955C mutation leads to an autosomal dominant phenotype, but the effects are delayed in onset. Although referred to as "early onset PEO", symptoms do not appear until the patients are in their thirties. Thus mtDNA replication is sufficient for individuals to live normally but then develop symptoms later in life. It is perhaps surprising that such drastic effects on the kinetics of incorporation are not lethal. Here we can only speculate about the relationship between the enzymology of pol γ and the physiological consequences of mutations. We presume that both mutant polymerase and a wild-type polymerase are present in the same mitochondrion, and perhaps mechanisms that regulate mtDNA copy number somehow compensate for the defective polymerase by allowing the wild-type enzyme to do most of the replication. Nonetheless, the drastic effects on catalytic efficiency and fidelity could ultimately cause the large deletions that are present in mtDNA in PEO patients by slowing the rate of replication such that replication stalls at long T tracks (16, 17, 39). More importantly perhaps, mutations introduced by the defective enzyme would be passed on to subsequent generations of mtDNA replication. Accordingly, it is possible that defects leading to reduced fidelity are more important than defects leading to slower rates of polymerization. While feedback mechanisms could compensate for reduced rates, mutations introduced during each round of replication accumulate over time and could explain the delayed onset of the disease. Increased mutation frequency can lead to defects in mtDNA that lead to increased oxidative stress, which in turn could lead to increased accumulation of defects in the mtDNA. The accumulation of mutations may result in more severe symptoms such as Parkinson's and premature ovarian failure (17, 29). The dual expression of both the Y955C and wild-type polymerase allow for some level of faithful replication and likely prevents all out failure of mtDNA replication.</p><p>A recent report noted phenomena suggesting that the replication complex stalls at A:T tracks (40). Our results provide quantitative data to support the proposed slower rate of replication at dATP:T base pairs. However, simple stalling of the replication complex may not produce the observed physiological effects if other regulatory mechanisms compensate for the reduced number of active replisomes. Alternatively, stalled intermediates could lead to the accumulation of deletions in the mtDNA. Further studies of the polymerase need to be done in the presence of the complete replisome to fully understand these dynamics.</p>