Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases

DNA binding properties of the Type 1 DNA polymerases from Thermus aquaticus (Taq, Klentaq) and Escherichia coli (Klenow) have been examined as a function of [KCl] and [MgCl(2)]. Full-length Taq and its Klentaq "large fragment" behave similarly in all assays. The two different species of polymerases bind DNA with sub-micromolar affinities in very different salt concentration ranges. Consequently, at similar [KCl] the binding of Klenow is approximately 3 kcal/mol (150x) tighter than that of Taq/Klentaq to the same DNA. Linkage analysis reveals a net release of 2-3 ions upon DNA binding of Taq/Klentaq and 4-5 ions upon binding of Klenow. DNA binding of Taq at a higher temperature (60 degrees C) slightly decreases the ion release. Linkage analysis of binding versus [MgCl(2)] reports the ultimate release of approximately 1 Mg(2+) ion upon complex formation. However, the MgCl(2) dependence for Klenow, but not Klentaq, shows two distinct phases. In 10 mm EDTA, both polymerase species still bind DNA, but their binding affinity is significantly diminished, Klenow more than Klentaq. In summary, the two polymerase species, when binding to identical DNA, differ substantially in their sensitivity to the salt concentration range, bind with very different affinities when compared under similar conditions, release different numbers of ions upon binding, and differ in their interactions with divalent cations.     

Extreme free energy of stabilization of Taq DNA polymerase

We have examined the chemical denaturations of the Klentaq and Klenow large-fragment domains of the Type 1 DNA polymerases from Thermus aquaticus (Klentaq) and Escherichia coli (Klenow) under identical solution conditions in order to directly compare the stabilization energetics of the two proteins. The high temperature stability of Taq DNA polymerase is common knowledge, and is the basis of its use in the polymerase chain reaction. This study, however, is aimed at understanding the thermodynamic basis for this high-temperature stability. Chemical denaturations with guanidine hydrochloride report a folding free energy (DeltaG) for Klentaq that is over 20 kcal/mol more favorable than that for Klenow under the conditions examined. This difference between the stabilization free energies of a homologous mesophilic-thermophilic protein pair is significantly larger than generally observed. This is due in part to the fact that the stabilization free energy for Klentaq polymerase, at 27.5 kcal/mol, is one of the largest ever determined for a monomeric protein. Large differences in the chemical midpoints of the unfolding (Cm) and the dependences of the unfolding free energy on denaturant concentration in the transition region (m-value) between the two proteins are also observed. Measurements of the sedimentation coefficients of the two proteins in the native and denatured states report that both proteins approximately double in hydrodynamic size upon denaturation, but that Klentaq expands somewhat more than Klenow.     

Thermodynamics of the DNA Structural Selectivity of the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the ΔC_p of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.     

Interactions of replication versus repair DNA substrates with the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus

Different DNA polymerases partition differently between replication and repair pathways. In this study we examine if two Pol I family polymerases from evolutionarily distant organisms also differ in their preferences for replication versus repair substrates. The DNA binding preferences of Klenow and Klentaq DNA polymerases, from Escherichia coli and Thermus aquaticus respectively, have been studied using a fluorescence competition binding assay. Klenow polymerase binds primed-template DNA (the replication substrate) with up to 50× higher affinity than it binds to nicked DNA, DNA with a 2 base single-stranded gap, blunt-ended DNA, or to a DNA end with a 3′ overhang. In contrast, Klentaq binds all of these DNAs almost identically, indicating that Klenow has a stronger ability to discriminate between replication and repair substrates than Klentaq. In contrast, both polymerases bind mismatched primed-template and blunt-ended DNA tighter than they bind matched primed-template DNA, suggesting that these two proteins may share a similar mechanism to identify mismatched DNA, despite the fact that Klentaq has no proofreading ability. In addition, the presence or absence of 5′- or 3′-phosphates has slightly different effects on DNA binding by the two polymerases, but again reinforce Klenow's more effective substrate discrimination capability.     

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.     

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.     

Synthesis of new modified DNAs by hyperthermophilic DNA polymerase: substrate and template specificity of functionalized thymidine analogues bearing an sp3-hybridized carbon at the C5 alpha-position for several DNA polymerases

Novel thymidine analogue triphosphates, which have an sp3-hybridized carbon at the C5 alpha-position with amino-linker arms, a methyl ester, or a carboxyl group at the C5 sidearm, were good substrates for primer-extension reactions by DNA polymerase from Pyrococcus kodakaraensis (KOD Dash DNA polymerase), yielding exclusively full-length products. The resulting modified DNA was further allowed to react with a functional molecule such as fluorescein isothiocyanate. By contrast, only truncated products were formed from the thymidine analogue substrate bearing the amino-linker arm or the negatively charged carboxyl group using Taq, Tth DNA polymerase, or DNA polymerase I from E. coli (Klenow fragment). The results indicate either that the thymidine analogue was not accepted by the enzymes, or that the polymerases could not extend the products, once the analogue had been incorporated, depending on the type of the analogue. A conventional thymidine analogue bearing an aminopropenyl group at the C5-position was accepted by all enzymes, among which KOD Dash DNA polymerase showed the highest activity for the polymerization with this analogue. Templates bearing the thymidine analogues in place of one thymidine residue were read by KOD Dash, Taq, Tth DNA polymerases, and the Klenow fragment giving the full-length product. KOD Dash DNA polymerase could expand structural diversities of substrates that can be used to prepare modified DNAs.     

Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.     

Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast

Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 3' flaps that can form during imprecise pairing. Indeed, a mutation in the 3'-5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ.     

Family A and family B DNA polymerases are structurally related: evolutionary implications.

In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.     

Building better polymerases: Engineering the replication of expanded genetic alphabets

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.     

Structure-specific DNA-induced conformational changes in Taq polymerase revealed by small angle neutron scattering

The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5'-nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNA-bound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.     

RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.     

The roles of Klenow processing and flap processing activities of DNA polymerase I in chromosome instability in Escherichia coli K12 strains.

The sequences of spontaneous mutations occurring in the endogenous tonB gene of Escherichia coli in the DeltapolA and polA107 mutant strains were compared. Five categories of mutations were found: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, (4) duplications, and (5) other mutations. The DeltapolA strain, which is deficient in both Klenow domain and 5' --> 3' exonuclease domain of DNA polymerase I, shows a marked increase in categories 1-4. The polA107 strain, which is deficient in the 5' --> 3' exonuclease domain but proficient in the Klenow domain, shows marked increases in categories 3 and 4 but not in 1 or 2. Previously, we reported that the polA1 strain, which is known to be deficient in the Klenow domain but proficient in the 5' --> 3' exonuclease domain, shows increases in categories 1 and 2 but not in 3 or 4. The 5' --> 3' exonuclease domain of DNA polymerase I is a homolog of the mammalian FEN1 and the yeast RAD27 flap nucleases. We therefore proposed the model that the Klenow domain can process deletion and minus frameshift mismatch in the nascent DNA and that flap nuclease can process plus frameshift and duplication mismatch in the nascent DNA.     

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.Escherichia coli DNA polymerase (DNAP)² I is a single subunit polymerase that is organized into three functional domains: an N-terminal domain that is associated with 5′ → 3′ exonuclease activity, an intermediate domain that carries the 3′ → 5′ proofreading activity, and a C-terminal domain that is associated with the 5′ → 3′ template-directed polymerization activity. An important role of DNAP I is to remove the RNA primers of the Okazaki fragments formed during lagging strand DNA synthesis in E. coli replication and to fill in the resulting gaps by template-directed DNA synthesis (1). An N-terminal deletion mutant of DNAP I, known as the “large fragment” or Klenow form of the enzyme, contains only the polymerase (pol) and the 3′ → 5′ exonuclease (exo) domains. The Klenow polymerase has served and continues to serve as an excellent model system for isolating and defining general structure-function relationships in polymerases and in the supporting machinery of DNA replication.The main function of the 3′ → 5′ exonuclease activity of DNAP I is to remove misincorporated nucleotide residues from the 3′-end of the primer (2), thus contributing significantly to the overall fidelity of DNA replication (3). Contrary to initial expectations, crystallographic studies showed that the pol and exo active sites are quite far apart in replication polymerases, about 30 Å in Klenow (4). As a consequence, the ability of polymerases to “shuttle” the 3′-end of the primer strand efficiently between the pol and the exo sites in order to rectify misincorporation events during polymerization is critical to maintaining the overall accuracy of template-directed replication. Elucidation of the mechanisms of this shuttling and determination of the factors that control the rates (and equilibria) of the active site switching reaction will certainly increase our understanding of fidelity control by DNA polymerases.An early crystallographic study of the Klenow polymerase complexed with fully paired primer-template (P/T) DNA revealed that 3–4 nt of the 3′-primer terminus had been unwound from the template stand and partitioned into the exo site and that an extended single-stranded DNA (ssDNA) binding pocket of the exo site appeared to make position-specific hydrophobic contacts with the unstacked bases at the 3′-end of the primer (4). A separate crystallographic study of an editing complex confirmed that an ssDNA fragment 4 nt in length was bound at the exo site in the same conformation as seen for the single-stranded 3′-primer sequence unwound from P/T DNA (5). A structure of Klenow polymerase with the DNA bound at the pol site has not yet been reported, although such structures have been obtained for other homologous polymerases, including Klentaq (the “large fragment” of Thermus aquaticus (Taq) DNAP), Bacillus stearothermophilus (Bst) “large fragment” polymerase, and the T7 DNAP (6–8), all of which are members of the polymerase family that includes Klenow.The amino acid residues involved in the binding of DNA at the pol site in these polymerases (determined from co-crystal structures) and those of Klenow (determined by site-directed mutagenesis studies (9, 10)) are highly conserved, suggesting that a similar DNA binding mode at the pol site may apply to all of the DNAP I polymerases. The crystal structure of Klenow revealed that the polymerization domain has a shape reminiscent of a right hand in which the palm, fingers, and thumb domains form the DNA-binding crevice. Structural studies with various DNAP I polymerases in the presence of P/T DNA constructs yielded an “open” binary complex, whereas the addition of the next correct dNTP (as a chain-terminating dideoxy-NTP) resulted in the formation of a catalytically competent “closed” ternary complex (6–8). In the latter complex, the 3′-primer terminus was base-paired with the template DNA, and the templating base was poised for incorporation of the next correct nucleotide. These structures showed that the conformation of the DNA primer terminus bound at the pol site is markedly different from that of the “frayed open” primer observed at the exo site in Klenow (4, 5).Although crystallographic studies have provided a wealth of information about the conformations of the DNA substrates bound at the active sites of DNAP, replication itself is a dynamic process (reviewed in Ref. 11), and it is critical to be able to distinguish between various forms of DNA-polymerase complexes in solution in order to fully understand the mechanistic details of the replication process. A solution approach used by Millar and co-workers (reviewed in Ref. 12) for studying the conformation of DNA in these complexes involved measuring the time-resolved fluorescence anisotropy properties of a dansyl fluorophore attached to a DNA base located 8 bp upstream of the P/T DNA junction. The changes in the lifetime of the fluorophore, which appeared to depend mostly on the local environment occupied by the probe within the protein (i.e. buried versus partially exposed), were correlated with specific binding conformations of the primer to provide an estimate of the fractional occupancy of the pol and the exo sites. Reha-Krantz and co-workers (13) more recently used a related approach, here involving the monitoring of changes in the fluorescent lifetimes of a single 2-aminopurine (2-AP) base (a fluorescent analogue of adenine) site-specifically substituted in the template strand at the P/T junction, to make similar fractional occupancy measurements. However, we note that structural interpretations of these fluorescence experiments relied heavily on the available crystal structures, and it remained to be shown directly that the 3′-end of the primer in P/T DNA constructs assumes the same distribution of conformations when bound to the protein in solution.To get around this problem, as well as to directly investigate the conformations of the primer DNA in both active sites of the Klenow and Klentaq polymerases, we have used a novel CD spectroscopic approach to characterize the solution conformations of primer DNA bound to Klenow and Klentaq DNAPs. Previously, we had shown that CD spectroscopy, in conjunction with fluorescence measurements, can be used to examine changes in local DNA and RNA conformations at 2-AP dimer probes inserted at specified positions within the nucleic acid frameworks of a variety of macromolecular machines functioning in solution (14–16). 2-AP is a structural isomer of adenine that forms base pairs with thymine in DNA (and uridine in RNA), and the substitution of 2-AP for adenine in such bp does not significantly perturb the structure or stability of the resultant double helix. Furthermore, when these probes are used as dimer pairs, the CD spectrum primarily reflects the interaction of the transition dipoles of the two probes themselves and thus the local conformation of the DNA at those positions within the P/T DNA. The characteristic CD and fluorescence signals for 2-AP probes in nucleic acids occur at wavelengths of >300 nm, a spectral region in which the protein and the canonical nucleic acid components of the “macromolecular machines of gene expression” are otherwise transparent. In this study, we have examined the binding of Klenow and Klentaq polymerases to P/T DNA constructs that were designed to be comparable with the nucleic acid components of functioning replication complexes. By examining the low energy CD spectra of site-specifically placed 2-AP probes, we have been able to characterize base conformations at defined positions within the DNA to reveal conformational features of specific DNA bases bound at and near both the pol and the exo active sites of these polymerases. These measurements, in that they directly reflect the actual conformations of the DNA chains bound within the active sites of the functioning polymerase, have also provided a direct means to estimate the equilibrium distributions of primer ends between the two active sites for various P/T DNA constructs.     

Crystal structure of an archaebacterial DNA polymerase.

BACKGROUND: Members of the Pol II family of DNA polymerases are responsible for chromosomal replication in eukaryotes, and carry out highly processive DNA replication when attached to ring-shaped processivity clamps. The sequences of Pol II polymerases are distinct from those of members of the well-studied Pol I family of DNA polymerases. The DNA polymerase from the archaebacterium Desulfurococcus strain Tok (D. Tok Pol) is a member of the Pol II family that retains catalytic activity at elevated temperatures. RESULTS: The crystal structure of D. Tok Pol has been determined at 2.4 A resolution. The architecture of this Pol II type DNA polymerase resembles that of the DNA polymerase from the bacteriophage RB69, with which it shares less than approximately 20% sequence identity. As in RB69, the central catalytic region of the DNA polymerase is located within the 'palm' subdomain and is strikingly similar in structure to the corresponding regions of Pol I type DNA polymerases. The structural scaffold that surrounds the catalytic core in D. Tok Pol is unrelated in structure to that of Pol I type polymerases. The 3'-5' proofreading exonuclease domain of D. Tok Pol resembles the corresponding domains of RB69 Pol and Pol I type DNA polymerases. The exonuclease domain in D. Tok Pol is located in the same position relative to the polymerase domain as seen in RB69, and on the opposite side of the palm subdomain compared to its location in Pol I type polymerases. The N-terminal domain of D. Tok Pol has structural similarity to RNA-binding domains. Sequence alignments suggest that this domain is conserved in the eukaryotic DNA polymerases delta and epsilon. CONCLUSIONS: The structure of D. Tok Pol confirms that the modes of binding of the template and extrusion of newly synthesized duplex DNA are likely to be similar in both Pol II and Pol I type DNA polymerases. However, the mechanism by which the newly synthesized product transits in and out of the proofreading exonuclease domain has to be quite different. The discovery of a domain that seems to be an RNA-binding module raises the possibility that Pol II family members interact with RNA.     

One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography

The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.     

Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).