Plasmid expression of Deinococcus radiodurans RecA confers UV-A protection to Escherichia coli with an inverse protein dose dependence, which does not exceed conspecific RecA protection.

Low level expression in Escherichia coli of the RecA protein from the radiation resistant bacterium Deinococcus radiodurans protects a RecA deficient strain of E. coli from UV-A irradiation by up to ∼160% over basal UV-A resistance. The protection effect is inverse protein dose dependent: increasing the expression level of the D. radiodurans RecA (DrRecA) protein decreases the protection factor. This inverse protein dose dependence effect helps resolve previously conflicting reports of whether DrRecA expression is protective or toxic for E. coli. In contrast to the D. radiodurans protein effect, conspecific plasmid expression of E. coli RecA protein in RecA deficient E. coli is consistently protective over several protein expression levels, as well as consistently more protective to higher levels of UV-A exposure than that provided by the D. radiodurans protein. The results indicate that plasmid expression of D. radiodurans RecA can modestly enhance the UV resistance of living E. coli, but that the heterospecific protein shifts from protective to toxic as expression is increased.

Pol I DNA polymerases stimulate DNA end-joining by Escherichia coli DNA ligase.

Klenow and Klentaq are the large fragment domains of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, respectively. Herein, we show that both polymerases can significantly stimulate complementary intermolecular end-joining ligations by E.coli DNA ligase when the polymerases are present at concentrations lower than that of the DNA substrates. In contrast, high polymerase concentrations relative to the DNA substrates inhibit the intermolecular ligation activity of DNA ligase. Neither polymerase was able to stimulate the DNA ligase from T4 bacteriophage. Additionally, nick-closure by E. coli DNA ligase (but not T4 ligase) is slightly stimulated by both polymerases, but only at about 10% of the magnitude seen for end-joining enhancement. The data represent one of the first observations of direct polymerase-ligase interactions in prokaryotes, and suggest that the polymerases stabilize the associated DNA ends during intermolecular ligation, and that such a complex can be taken advantage of by some, but not all, DNA ligases.

Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics.

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs-Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔCp ) of folding. If increased or enhanced non-covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow-Klentaq homologous pair, the folding enthalpy (ΔHfold ) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (ΔGfold ) originates from a significantly reduced entropic penalty of folding (ΔSfold ). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic-thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar "reduced ΔCp mechanism" for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced-entropic-penalty model.

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 (ΔHbinding) crosses zero (TH) and becomes favorable (negative). This TH/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.

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.

Analysis of free energy versus temperature curves in protein folding and macromolecular interactions.

Plots of free energy versus temperature are commonly called stability curves or Gibbs-Helmholtz curves, and they have proven to be extremely useful in protein folding and ligand-binding studies. Curvature in a Gibbs-Helmholtz or stability plot is indicative of a heat capacity change, and some of their primary uses in biochemistry over the past few decades have included determining ΔCp values and comparing ΔCp values between two related processes. This chapter describes basic approaches for analyzing curved Gibbs-Helmholtz plots, along with two specific extensions of standard Gibbs-Helmholtz plot analysis: (1) translating ΔG of folding versus temperature into ΔH and ΔS versus temperature for comparing mesophilic-thermophilic protein pairs, and (2) fitting Gibbs-Helmholtz plots to determine if ΔCp changes with temperature or not. Neither of these extensions is new, but they are infrequently used, and their use is particularly germane to certain molecular interpretations of thermodynamic information from ΔG versus temperature curves. It is shown that translating ΔG of folding into ΔH and ΔS of folding versus temperature for a mesophilic-thermophilic protein pair can immediately influence possible structural hypotheses for thermal stabilization of thermophilic proteins. It is also shown that very small temperature-dependent heat capacity changes (ΔΔCp values) can be obtained from extended fits to ΔG versus temperature plots, and that these very small ΔΔCp values can have serious consequences for any attempt to correlate ΔCp with ΔASA for some reactions.

The glutamate effect on DNA binding by pol I DNA polymerases: osmotic stress and the effective reversal of salt linkage.

The significant enhancing effect of glutamate on DNA binding by Escherichia coli nucleic acid binding proteins has been extensively documented. Glutamate has also often been observed to reduce the apparent linked ion release (Deltan(ions)) upon DNA binding. In this study, it is shown that the Klenow and Klentaq large fragments of the Type I DNA polymerases from E. coli and Thermus aquaticus both display enhanced DNA binding affinity in the presence of glutamate versus chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow displays an apparently decreased Deltan(ions) in the presence of Kglutamate, while Klentaq appears not to display an anion-specific effect on Deltan(ions). Osmotic stress experiments reveal that DNA binding by Klenow and Klentaq is associated with the release of approximately 500 to 600 waters in the presence of KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the osmotic-stress-measured hydration change associated with DNA binding (to approximately 150-200 waters released), suggesting that glutamate plays a significant osmotic role. Measurements of the salt-DNA binding linkages were extended up to 2.5 M Kglutamate to further examine this osmotic effect of glutamate, and it is observed that a reversal of the salt linkage occurs above 800 mM for both Klenow and Klentaq. Salt-addition titrations confirm that an increase of [Kglutamate] beyond 1 M results in rebinding of salt-displaced polymerase to DNA. These data represent a rare documentation of a reversed ion linkage for a protein-DNA interaction (i.e., enhanced binding as salt concentration increases). Nonlinear linkage analysis indicates that this unusual behavior can be quantitatively accounted for by a shifting balance of ionic and osmotic effects as [Kglutamate] is increased. These results are predicted to be general for protein-DNA interactions in glutamate salts.

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 Delta C(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.

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.

Prevalence of temperature-dependent heat capacity changes in protein-DNA interactions.

A large, negative DeltaCp of DNA binding is a thermodynamic property of the majority of sequence-specific DNA-protein interactions, and a common, but not universal property of non-sequence-specific DNA binding. In a recent study of the binding of Taq polymerase to DNA, we showed that both the full-length polymerase and its "Klentaq" large fragment bind to primed-template DNA with significant negative heat capacities. Herein, we have extended this analysis by analyzing this data for temperature-variable heat capacity effects (DeltaDeltaCp), and have similarly analyzed an additional 47 protein-DNA binding pairs from the scientific literature. Over half of the systems examined can be easily fit to a function that includes a DeltaDeltaCp parameter. Of these, 90% display negative DeltaDeltaCp values, with the result that the DeltaCp of DNA binding will become more negative with rising temperature. The results of this collective analysis have potentially significant consequences for current quantitative theories relating DeltaCp values to changes in accessible surface area, which rely on the assumption of temperature invariance of the DeltaCp of binding. Solution structural data for Klentaq polymerase demonstrate that the observed heat capacity effects are not the result of a coupled folding event.

Applications of fluorescence anisotropy to the study of protein-DNA interactions.

The use of fluorescence anisotropy to monitor protein-DNA interactions has been on the rise since its introduction by Heyduk and Lee in 1990. As a solution-based, true-equilibrium, real-time method, it has several advantages (and a few disadvantages) relative to the more classical methods of filter binding and the electrophoretic mobility shift assay (gel shift). This chapter discusses the basis for monitoring protein-DNA interactions using fluorescence anisotropy, as well as the advantages and disadvantages of the method, but the bulk of the chapter is devoted to experimental tips and guidance meant to augment existing reviews of the method. The focus is on the current primary use of the method: direct measurement of binding isotherms for protein-DNA interactions in vitro. A short summary of emerging applications of the method is also included.

Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration.

The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 degrees C, from 40 to 62 degrees C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH4+>Na+>Li+>K+. Of the divalent cations tested: Mg+2 and Mn+2 significantly stabilize the protein, while Ni+2 dramatically destabilizes the protein. Stability measurements performed in combined Mg+2 plus Na+ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein.

Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA.

DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA. The temperature dependence of the binding affinity exhibits distinct curvature, with tightest binding at 25-30 degrees C. Nonlinear temperature dependence indicates Klenow binds different primed-template constructs with large heat capacity (DeltaCp) values (-870 to -1220 cal/mole K) and thus exhibits large temperature dependent changes in enthalpy and entropy. Binding is entropy driven at lower temperatures and enthalpy driven at physiological temperatures. Large negative DeltaCp values have been proposed to be a 'signature' of site-specific DNA binding, but type I DNA polymerases do not exhibit significant DNA sequence specificity. We suggest that the binding of Klenow to a specific DNA structure, the primed-template junction, results in a correlated thermodynamic profile that mirrors what is commonly seen for DNA sequence-specific binding proteins. Klenow joins a small number of other DNA-sequence independent DNA binding proteins which exhibit unexpectedly large negative DeltaCp values. Spectroscopic measurements show small conformational rearrangements of both the DNA and Klenow upon binding, and small angle x-ray scattering shows a global induced fit conformational compaction of the protein upon binding. Calculations from both crystal structure and solution structural data indicate that Klenow DNA binding is an exception to the often observed correlation between DeltaCp and changes in accessible surface area. In the case of Klenow, surface area burial can account for only about half of the DeltaCp of binding.

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 binding of Thermus aquaticus DNA polymerase to primed-template DNA.

DNA binding of the Type 1 DNA polymerase from Thermus aquaticus (Taq polymerase) and its Klentaq large fragment domain have been studied as a function of temperature. Equilibrium binding assays were performed from 5 to 70 degrees C using a fluorescence anisotropy assay and from 10 to 60 degrees C using isothermal titration calorimetry. In contrast to the usual behavior of thermophilic proteins at low temperatures, Taq and Klentaq bind DNA with high affinity at temperatures down to 5 degrees C. The affinity is maximal at 40-50 degrees C. The DeltaH and DeltaS of binding are highly temperature dependent, and the DeltaCp of binding is -0.7 to -0.8 kcal/mol K, for both Taq and Klentaq, with good agreement between van't Hoff and calorimetric values. Such a thermodynamic profile, however, is generally associated with sequence-specific DNA binding and not non- specific binding. Circular dichroism spectra show conformational rearrangements of both the DNA and the protein upon binding. The high DeltaCp of Taq/Klentaq DNA binding may be correlated with structure-specific binding in analogy to sequence- specific binding, or may be a general characteristic of proteins that primarily bind non-specifically to DNA. The low temperature DNA binding of Taq/Klentaq is suggested to be a general characteristic of thermophilic DNA binding proteins.

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins.

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases.

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain. Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower T(m) values, but almost identical van't Hoff enthalpy Delta H values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

Global conformations, hydrodynamics, and X-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution.

Escherichia coli polymerase 1 (Pol 1) and Thermus aquaticus Taq polymerase are homologous Type I DNA polymerases, each comprised of a polymerase domain, a proofreading domain (inactive in Taq), and a 5' nuclease domain. "Klenow" and "Klentaq" are the large fragments of Pol 1 and Taq and are functional polymerases lacking the 5' nuclease domain. In the available crystal structures of full-length Taq, the 5' nuclease domain is positioned in two different orientations: in one structure, it is extended out into solution, whereas in the other, it is folded up against the polymerase domain in a more compact structure. Analytical ultracentrifugation experiments report s20,w values of 5.05 for Taq, 4.1 for Klentaq, 5.3 for E. coli Pol 1, and 4.6 for Klenow. Measured partial specific volumes are all quite similar, indicating no significant differences in packing density between the mesophilic and thermophilic proteins. Small angle x-ray scattering studies report radii of gyration of 38.3 A for Taq, 30.7 A for Klentaq, and 30.5 A for Klenow. The hydrodynamic and x-ray scattering properties of the polymerases were also calculated directly from the different crystal structures using the programs HYDROPRO (Garcia De La Torre, J., Huertas, M. L., and Carrasco, B. (2000) Biophys J. 78, 719-730) and CRYSOL (Svergun, D. I., Barberato, C., and Koch, M. H. J. (1995) J. Appl. Crystalogr. 28, 768-773), respectively. The combined experimental and computational characterizations indicate that the full-length polymerases in solution are in a conformation where the 5' nuclease domain is extended into solution. Further, the radius of gyration, and hence the global conformation of Taq polymerase, is not altered by the binding of either matched primer template DNA or ddATP.

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.