Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 10⁵, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.     

DNA polymerase activity at the single-molecule level

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3'→5' exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase-DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA-protein complexes.     

DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions

DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.     

Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.     

Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy

Prior to undergoing postsynthetic 3'-5' editing (proofreading), a defective DNA primer terminus must be transferred from the 5'-3' polymerase active site to a remote 3'-5' exonuclease site. To elucidate the mechanisms by which this occurs, we have used time-resolved fluorescence spectroscopy to study the interaction of dansyl-labeled DNA primer/templates with the Klenow fragment of Escherichia coli DNA polymerase I. The dansyl probe is positioned such that when the DNA substrate occupies the polymerase active site, the probe is solvent-exposed and possesses a short average fluorescence lifetime (4.7 ns) and extensive angular diffusion (42.5 degrees). Conversely, when the DNA substrate occupies the exonuclease active site, the probe becomes buried within the protein, resulting in an increase in the average lifetime (14.1 ns) and a decrease in the degree of angular diffusion (14.4 degrees ). If both polymerase and exonuclease binding modes are populated (lower limit approximately 5%), their markedly different fluorescence properties cause the anisotropy to decay with a characteristic "dip and rise" shape. Nonlinear least-squares analysis of these data recovers the ground-state mole fractions of exposed (x(e)) and buried (x(b)) probes, which are equivalent to the equilibrium proportions of the DNA substrate bound at the polymerase and exonuclease sites, respectively. The distribution between the polymerase and exonuclease binding modes is given by the equilibrium partitioning constant K(pe) (equal to x(b)/x(e)). The important determinants of the proofreading process can therefore be identified by changes made to either the protein or DNA that perturb the partitioning equilibrium and hence alter the magnitude of K(pe).     

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.     

Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.     

Molecular events during translocation and proofreading extracted from 200 static structures of DNA polymerase

DNA polymerases in family B are workhorses of DNA replication that carry out the bulk of the job at a high speed with high accuracy. A polymerase in this family relies on a built-in exonuclease for proofreading. It has not been observed at the atomic resolution how the polymerase advances one nucleotide space on the DNA template strand after a correct nucleotide is incorporated, that is, a process known as translocation. It is even more puzzling how translocation is avoided after the primer strand is excised by the exonuclease and returned back to the polymerase active site once an error occurs. The structural events along the bifurcate pathways of translocation and proofreading have been unwittingly captured by hundreds of structures in Protein Data Bank. This study analyzes all available structures of a representative member in family B and reveals the orchestrated event sequence during translocation and proofreading.     

Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I

A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I). Escherichia coli DNA pol I bypassed the lesion efficiently, but partial inhibition was observed for T4 DNA polymerase. The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center. These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase–DNA–dNTP complex. When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion. The replication block was overcome by higher dNTP concentrations. Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks.     

A possible mechanism for the dynamics of transition between polymerase and exonuclease sites in a high-fidelity DNA polymerase

The fidelity of DNA synthesis by DNA polymerase is significantly increased by a mechanism of proofreading that is performed at the exonuclease active site separate from the polymerase active site. Thus, the transition of DNA between the two active sites is an important activity of DNA polymerase. Here, based on our proposed model, the rates of DNA transition between the two active sites are theoretically studied. With the relevant parameters, which are determined from the available crystal structure and other experimental data, the calculated transfer rate of correctly base-paired DNA from the polymerase to exonuclease sites and the transfer rate after incorporation of a mismatched base are in good agreement with the available experimental data. The transfer rates in the presence of two and three mismatched bases are also consistent with the previous experimental data. In addition, the calculated transfer rate from the exonuclease to polymerase sites has a large value even with the high binding affinity of 3′-5′ ssDNA for the exonuclease site, which is also consistent with the available experimental value. Moreover, we also give some predictive results for the transfer rate of DNA containing only A:T base pairs and that of DNA containing only G:C base pairs.     

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3′→5′ exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.     

Kinetics and fidelity of polymerization by DNA polymerase III from Sulfolobus solfataricus

We have biochemically and kinetically characterized the polymerase and exonuclease activities of the third B-family polymerase (Dpo3) from the hyperthermophilic Crenarchaeon, Sulfolobus solfataricus (Sso). We have established through mutagenesis that despite incomplete sequence conservation, the polymerase and exonuclease active sites are functionally conserved in Dpo3. Using pre-steady-state kinetics, we can measure the fidelity of nucleotide incorporation by Dpo3 from the polymerase active site alone to be 10(3)-10(4) at 37 °C. The functional exonuclease proofreading active site will increase fidelity by at least 10(2), making Dpo3 comparable to other DNA polymerases in this family. Additionally, Dpo3's exonuclease activity is modulated by temperature, where a loss of promiscuous degradation activity can be attributed to a reorganization of the exonuclease domain when it is bound to primer-template DNA at high temperatures. Unexpectedly, the DNA binding affinity is weak compared with those of other DNA polymerases of this family. A comparison of the fidelity, polymerization kinetics, and associated functional exonuclease domain with those previously reported for other Sso polymerases (Dpo1 and Dpo4) illustrates that Dpo3 is a potential player in the proper maintenance of the archaeal genome.     

Proofreading exonuclease activity of human DNA polymerase δ and its effects on lesion-bypass DNA synthesis

Replicative DNA polymerases possess 3′ → 5′ exonuclease activity to reduce misincorporation of incorrect nucleotides by proofreading during replication. To examine if this proofreading activity modulates DNA synthesis of damaged templates, we constructed a series of recombinant human DNA polymerase δ (Pol δ) in which one or two of the three conserved Asp residues in the exonuclease domain are mutated, and compared their properties with that of the wild-type enzyme. While all the mutant enzymes lost more than 95% exonuclease activity and severely decreased the proofreading activity than the wild-type, the bypass efficiency of damaged templates was varied: two mutant enzymes, D515V and D402A/D515A, gave higher bypass efficiencies on templates containing an abasic site, but another mutant, D316N/D515A, showed a lower bypass efficiency than the wild-type. All the enzymes including the wild-type inserted an adenine opposite the abasic site, whereas these enzymes inserted cytosine and adenine opposite an 8-oxoguanine with a ratio of 6:4. These results indicate that the exonuclease activity of human Pol δ modulates its intrinsic bypass efficiency on the damaged template, but does not affect the choice of nucleotide to be inserted.     

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.     

The proofreading exonuclease subunit epsilon of Escherichia coli DNA polymerase III is tethered to the polymerase subunit alpha via a flexible linker

Escherichia coli DNA polymerase III holoenzyme is composed of 10 different subunits linked by noncovalent interactions. The polymerase activity resides in the α-subunit. The ε-subunit, which contains the proofreading exonuclease site within its N-terminal 185 residues, binds to α via a segment of 57 additional C-terminal residues, and also to θ, whose function is less well defined. The present study shows that θ greatly enhances the solubility of ε during cell-free synthesis. In addition, synthesis of ε in the presence of θ and α resulted in a soluble ternary complex that could readily be purified and analyzed by NMR spectroscopy. Cell-free synthesis of ε from PCR-amplified DNA coupled with site-directed mutagenesis and selective ¹⁵N-labeling provided site-specific assignments of NMR resonances of ε that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of ε is connected to α via a flexible linker peptide comprising over 20 residues. This distinguishes the α : ε complex from other proofreading polymerases, which have a more rigid multidomain structure.     

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.     

Nucleotide excision by E. coli DNA polymerase I in proofreading and non-proofreading modes

Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms. When it binds to a template, the proofreading activity is usually switched off. As the enzyme progresses along the template, it becomes more and more competent for excision. This phenomenon introduces a link between fidelity and processivity. Processivity is best studied when the chain-length distributions of synthesized polymers are stationary. Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme. When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed. It seems that the polymerase starts by hydrolyzing the primer down to a length of 7–10 nucleotides and only then starts to add nucleotides. It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading. The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3′ → 5′ exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.     

DNA polymerase gamma from Xenopus laevis. II. A 3'----5' exonuclease is tightly associated with the DNA polymerase activity

Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease. The purified enzyme lacks 5'----3' exonuclease and endonuclease activity. The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures. In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol. The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function. The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.     

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.