The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data.

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role.     

A preliminary CD and NMR study of the Escherichia coli DNA polymerase III theta subunit.

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: alpha, epsilon, and theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of undetermined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of secondary structure: approximately 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, theta yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1H-15N HSQC analysis of uniformly-15N-labeled theta supports the conclusion that approximately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1H-15N HSQC spectra resulted when [U-15N]-theta was examined in the presence of epsilon186--the N-terminal domain of the epsilon-subunit. Further work is currently in progress to define the solution structure of theta and the theta-epsilon186 complex.     

Two functional domains of the epsilon subunit of DNA polymerase III

The epsilon subunit is the 3'-->5' proofreading exonuclease that associates with the alpha and theta subunits in the E. coli DNA polymerase III. Two fragments of the epsilon protein were prepared, and binding of these epsilon fragments with alpha and theta was investigated using gel filtration chromatography and exonuclease stimulation assays. The N-terminal fragment of epsilon, containing amino acids 2-186 (epsilon186), is a relatively protease-resistant core domain of the exonuclease. The purified recombinant epsilon186 protein catalyzes the cleavage of 3' terminal nucleotides, demonstrating that the exonuclease domain of epsilon is present in the N-terminal region of the protein. The absence of the C-terminal 57 amino acids of epsilon in the epsilon186 protein reduces the binding affinity of epsilon186 for alpha by at least 400-fold relative to the binding affinity of epsilon for alpha. In addition, stimulation of the epsilon186 exonuclease by alpha using a partial duplex DNA is about 50-fold lower than stimulation of the epsilon exonuclease by alpha. These results indicate that the C-terminal region of epsilon is required in the epsilonalpha association. To directly demonstrate that the C-terminal region of epsilon contains the alpha-association domain fusion protein, constructs containing the maltose-binding protein (MBP) and fragments of the C-terminal region of epsilon were prepared. Gel filtration analysis demonstrates that the alpha-association domain of epsilon is contained within the C-terminal 40 amino acids of epsilon. Also, the epsilon186 protein forms a tight complex with theta, demonstrating that the association of theta with epsilon is localized to the N-terminal region of epsilon. Association of epsilon186 and theta is further supported by the stimulation of the epsilon186 exonuclease in the presence of theta. These data support the concept that epsilon contains a catalytic domain located within the N-terminal region and an alpha-association domain located within the C-terminal region of the protein.     

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.     

Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.     

Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.     

The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit

The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta. Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta. Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit. The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta. theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.     

Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (epsilon) subunit of Escherichia coli DNA polymerase III

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH <6.5 and 400 min(-1) at pH >9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.     

A coproofreading Zn(2+)-dependent exonuclease within a bacterial replicase

The proofreading exonucleases of all DNA replicases contain acidic residues that chelate two Mg(2+) ions that participate in catalysis. DNA polymerase III holoenzymes contain their proofreading activity in a separate subunit, epsilon, which binds the polymerase subunit, alpha, through alpha's N-terminal php domain. Here we demonstrate that the alpha php domain contains a novel Zn(2+)-dependent 3' --> 5' exonuclease that preferentially removes mispaired nucleotides, providing the first example of a coediting nuclease.     

holE, the gene coding for the theta subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (epsilon-subunit) mutant.

DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an alpha epsilon theta heterotrimer that incorporates both a polymerase subunit (alpha; dnaE) and a proofreading subunit (epsilon; dnaQ). The role of theta is unknown. Here, we describe a null mutation of holE, the gene for theta. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting alpha which was identical to a suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the theta subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of epsilon mutants cannot be explained on the basis of interference with theta function.     

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.     

The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities

The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.     

NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta). The theta subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon.     

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

3'-->5'-exonucleases of the rat liver and the correction of replication errors]

Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes.     

Structural basis for proofreading during replication of the Escherichia coli chromosome

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.     

Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III

DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.     

Mutator and antimutator effects of the bacteriophage P1 hot gene product

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III theta subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III epsilon proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of theta. Experiments using chimeric theta/Hot proteins containing various domains of Hot and theta along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the epsilon subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.     

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.