©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Deoxynucleoside Triphosphate and Pyrophosphate Binding Sites in the Catalytically Competent Ternary Complex for the Polymerase Reaction Catalyzed by DNA Polymerase I (Klenow Fragment) (*)

(Received for publication, September 27, 1994)

Mekbib Astatke Nigel D. F. Grindley Catherine M. Joyce (§)

From the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have employed site-directed mutagenesis to identify those amino acid residues that interact with the deoxynucleoside triphosphate (dNTP) and pyrophosphate in the Klenow fragment-DNA-dNTP ternary complex. Earlier structural, mutagenesis, and labeling studies have suggested that the incoming dNTP molecule contacts a region on one side of the polymerase cleft, primarily involving residues within the so-called ``fingers'' subdomain. We have made mutations in residues seen to be close to the dNTP in the crystal structure of the Klenow fragment-dNTP binary complex and have examined their kinetic parameters, particularly K. The results are consistent with the notion that there are significant differences between the dNTP interactions in the binary and ternary complexes, although some contacts may be present in both. When dTTP is the incoming nucleotide, the side chains of Arg and Phe make the largest contributions to binding; measurement of K suggests that Arg contacts the beta- or -phosphate of the dNTP. With dGTP, the contribution of Arg remains the same, but the additional interactions are provided by both Lys and Phe, suggesting that the binding of the incoming dNTP is not identical under all circumstances. Mutations in Arg and Lys also cause a substantial decrease in the rate of polymerase-catalyzed incorporation, and sulfur elemental effect measurements indicate that loss of Arg (and perhaps also Lys) slows the rate of the chemical step of the reaction. Mutations of Arg, His, and Tyr affect the binding of DNA, suggesting that these mutations, whose effect on dNTP binding is small, may influence dNTP binding indirectly via the positioning of the DNA template-primer.


INTRODUCTION

The Klenow fragment of Escherichia coli DNA polymerase I contains the polymerase and 3`-5` (proofreading) exonuclease active sites of the parent molecule. Structural and biochemical studies have revealed that these two catalytic sites are located on two distinct structural domains(1, 2) . Klenow fragment was the first DNA synthesizing enzyme with a known three-dimensional structure(1) . Extensive mutagenesis and kinetic studies have combined with this structural data to provide a wealth of information on Klenow fragment, making it an ideal model to study the molecular mechanism of template-directed DNA synthesis (e.g.(3, 4, 5, 6, 7, 8) ; reviewed in (9) ). Protein sequence alignments (10) and examination of the four known polymerase structures, Klenow fragment(1) , human immunodeficiency virus-1 reverse transcriptase(11) , T7 RNA polymerase (12) , and DNA polymerase beta(13, 14) , suggest that even distantly related polymerases may have analogous active sites with a small number of crucial side chains in common. As a consequence, structure-function studies pursued on a simple enzyme system such as Klenow fragment may well be applicable to other more complex replication enzymes.

The molecular mechanism of the 3`-5` exonuclease reaction has been well characterized thanks to the structural data for complexes of Klenow fragment with deoxynucleoside monophosphate and with single-stranded DNA bound in the editing mode(1, 15, 16) , and to extensive mutagenesis studies(3, 17) . However, since the three-dimensional structure of the catalytically competent ternary complex for the polymerase reaction (Klenow fragment-duplex DNA-dNTP) (^1)is not known, the reaction mechanism at the polymerase active site is not as fully understood, although a plausible mechanism has gained widespread acceptance(18) . Comparison of the four known polymerase structures shows that the polymerase domain in each case contains a deep cleft, whose overall shape has been compared to that of a half-open right hand(11) , with the beta-sheet that forms the base of the cleft described as the ``palm,'' and the two subdomains that form the walls of the cleft described as ``fingers'' and ``thumb'' (Fig. 1). Protein sequence alignments (10) and mutagenesis data (reviewed in (9) ) indicate that the polymerase active site is located primarily on the palm subdomain. The most important active site residues appear to be a group of two or (more commonly) three carboxylates: Asp, Asp, and Glu in Klenow fragment. It has been proposed that these carboxylates act to coordinate a pair of divalent metal ions which then catalyze the phosphoryl transfer by a mechanism analogous to that used at the 3`-5` exonuclease site(9, 18) ; this mechanism is convincingly supported by the recent structural data for mammalian DNA polymerase beta(13, 19) . Kinetic studies have shown that, prior to the phosphoryl transfer, the ternary complex must undergo a nonchemical transformation, described as a conformational change(6, 8) ; the precise nature of this step is unclear at present.


Figure 1: Positions of amino acid residues that were mutated. The alpha-carbon backbone of Klenow fragment (1, 9) is shown schematically with alpha-helices represented by spiral ribbons and beta-strands by arrows. The 3`-5` exonuclease domain, in a slightly darker shade of gray, occupies the foreground. The polymerase domain is viewed down the large cleft, with the palm subdomain at the base, the fingers to the left and the thumb to the right. With the exception of Asp and Asp, which serve to mark the polymerase active site, the side chains that are illustrated correspond to those mutated in this study.



To understand fully the polymerase reaction catalyzed by Klenow fragment, the amino acid side chains that make contact with the primer-template and the incoming dNTP in the ternary complex must also be identified, and the role (if any) of these contact residues as the reaction proceeds must be determined. A variety of structural, chemical cross-linking, and mutagenesis studies have addressed the location of the dNTP binding site. Although these studies are generally in agreement in indicating a dNTP binding region that is largely on the fingers subdomain, encompassing the exposed face of Helix O (residues Arg to Tyr) and neighboring portions closer to the palm subdomain (Fig. 1), none of the experimental approaches allows an unambiguous interpretation of the data. The crystallographic studies (20) and the majority of the chemical cross-linking experiments were carried out on the binary Klenow fragment-dNTP complex; however, this complex is not catalytically competent since there is a requirement for the binding of the template-primer before dNTP can bind productively(21, 22) . Clearly, data obtained from studies of the binary complex will be of value in identifying catalytically relevant interactions only to the extent that particular interactions may be common to both binary and ternary complexes. Beese et al. (20) have made the entirely reasonable suggestion that the phosphate positions in the binary complex (near the positively-charged side chains of Lys and Arg) are more likely to be relevant to the ternary complex than are the base and sugar positions. In the binary complex the deoxyribose was seen to be close to Phe, while the position of the base varied in different experiments, presumably due to the absence of interactions with the opposing template base that would serve to anchor the dNTP in the enzyme-DNA-dNTP ternary complex.

As indicated above, most of the chemical cross-linking experiments that have been reported for Klenow fragment were carried out on binary complexes, so that, for example, experiments indicating that the nucleotide base is close to Tyr or His are subject to the same caveat as the structural data, namely that the position of the base moiety in the binary complex may be misleading(23, 24) . A more convincing labeling experiment, using 5`-fluorosulfonylbenzoyl deoxyadenosine as an affinity label in the presence of primer-template DNA, in order to mimic the ternary complex, suggested that the dNTP phosphates are close to the side chain of Arg(25) . Somewhat harder to classify is the labeling of Lys by pyridoxal phosphate(26) , a reagent claimed to be directed to the dNTP binding site. In this experiment dNTP, when bound either as a binary or a ternary complex, protected Lys from modification. The pyridoxal phosphate experiment illustrates a general weakness of the labeling approach, namely that some reagents may show specificity for certain types of side chains, so that labeling is not necessarily diagnostic for the closest side chain, but rather for a side chain of a particular chemical class that is within reasonable proximity to the reagent.

In the absence of structural data for the ternary complex, site-directed mutagenesis and measurement of the effect of the resulting mutations on K offers considerable advantages over the approaches already described because it probes the enzyme-DNA-dNTP ternary complex. The weakness of this approach is that it may be difficult to distinguish between mutations that exert an effect on dNTP binding due to loss of a dNTP contact residue and those whose effect is indirect via a change in position of the opposing DNA template base at the dNTP binding site. Thus far our mutational studies have identified 5 residues (Asp, Glu, Tyr, Arg, and Asn) whose mutation causes a weakening in dNTP binding, as measured by an increase in K(4, 5) . Since none of the effects were quantitatively large, and since several of these residues (particularly Tyr, Arg, and Asn) are in a region of the protein likely to be close to the template strand(27) , it is possible that our experiments to date have not identified any important direct contacts to the dNTP molecule in the ternary complex. To continue this investigation, we describe here a further series of mutations in residues that point into the polymerase cleft within the likely dNTP contact region (Fig. 1).


EXPERIMENTAL PROCEDURES

Materials

Poly(dA) and poly(dC) with average lengths of 300-500 residues, oligo(dT), oligo(dT)(14), ultrapure dNTPs, and dTTPalphaS (a mixture of Sp and Rp diastereomers) (^2)were purchased from Pharmacia LKB Biotech Inc. Radiolabeled nucleotides were purchased from Amersham Corp. or DuPont NEN. The 68-mer hairpin oligonucleotide, 5`-d(GTGTACGTATGATCATGCAGGTAGCCGATGAACTGGTCGAAAGACCAGTTCATCGGCTACCTGCATGA)-3` (see Fig. 4a), and oligo(dG) were synthesized by the Keck Biotechnology Resource Laboratory at Yale Medical School. Concentrations of radiolabeled oligonucleotides were estimated using a DNA dipstick quantitation kit supplied by Invitrogen. DNase I was from Cooper Biomedical. T(4) polynucleotide kinase was from New England Biolabs. Inorganic pyrophosphatase was purchased from Boehringer Mannheim as an ammonium sulfate slurry; it was prepared for use by spinning 10 µl of the slurry in a microcentrifuge for 3 min at 4 °C and dissolving the precipitate in 66 µl of 50 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, giving a working stock of 30 units/µl.


Figure 4: Measurement of K(DNA) using a gel mobility shift assay. A, the 68-mer hairpin oligonucleotide. This molecule, labeled at the 5` end with P, was employed as the substrate in the mobility shift assay. B, gel assay of the binding of the Q708A mutant protein to the 68-mer, carried out as described under ``Experimental Procedures.'' The protein concentration (in nM) increases from lane to lane in 2-fold steps from left to right (not all lanes are labeled). U indicates the mobility of the unbound 68-mer; A and B indicate the positions of the two protein-bound species that we have detected. C, a plot of the binding data obtained for the Q708A (experiment shown in panel B) and R682A mutant proteins. The fraction of the DNA substrate that is enzyme-bound is plotted as a function of protein concentration (bullet, Q708A; circle, R682A). By interpolation, K was calculated to be 1.4 nM for Q708A and 4.7 nM for R682A.



Production of Mutant Derivatives of Klenow Fragment

Mutations were made by oligonucleotide-directed mutagenesis on a uracil-containing M13 template containing the region of the polA gene that encodes the polymerase domain. The isolation, sequencing, and subcloning of the mutations into a high level expression plasmid, which also contained the D424A (3`-5` exonuclease-deficient) mutation, were all as described previously(4, 5) . Klenow fragment derivatives were overexpressed and purified by fast protein liquid chromatography (Pharmacia LKB) as described in detail elsewhere(28) . Peak fractions from the final column were mixed with an equal volume of glycerol and stored at -20 °C; the final buffer concentration was 50 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 50% (v/v) glycerol. The concentration of Klenow fragment was measured by the Bradford colorimetric assay (29) using the reagent supplied by Bio-Rad. A solution of bovine serum albumin of known concentration was used as a standard since we had previously shown that it gave the same result as a Klenow fragment standard.

Melting Temperature Studies

A solution of wild-type or mutant Klenow fragment (at about 4 µM in 20 mM potassium phosphate, pH 7.0, 15 mM NaCl, 15% (v/v) glycerol) in a 2-mm path length quartz cuvette (110-QS, Hellma) was placed in a thermostated block in a CD spectrophotometer (Aviv Model 62DS, Lakewood, NJ). Ellipticity measurements were taken at 222 nm as a function of temperature over the range 20-90 °C in 1 °C increments (samples were equilibrated for 2 min at each temperature). For each measurement, data values were averaged for 60 s and were smoothed over 10 points using the Aviv software. The thermal denaturation profile was corrected by subtraction at both the upper and lower baselines. The T(m) value (the temperature at which the protein is 50% unfolded) was determined by interpolation. The values given in Table 1are from a single determination for each protein.



Steady-state Kinetic Measurements of the Polymerase Reaction

The steady-state kinetic parameters, K(m) and k, for wild-type and mutant Klenow fragment derivatives were measured essentially as described by Polesky et al.(4) , using a homopolymeric poly(dA)-oligo(dT) substrate. The DNA substrate was made by annealing (dT) to poly(dA) at a molar ratio of 1 (dT) per 250-500 template A residues. For each determination, a range of 8-10 dTTP concentrations, which bracketed the K(m) value, was used and duplicate measurements were made at each dTTP concentration. The concentration of the DNA substrate was 2.4 µM (in primer termini); for each Klenow fragment derivative, the reaction rate was shown to be identical, within experimental error, for primer concentrations of 2.4 and 4 µM, confirming that the DNA concentration of 2.4 µM was saturating. The concentration of enzyme in the reaction mixture ranged from around 1 nM for wild-type Klenow fragment to around 40 nM for the least active mutant proteins. An improvement over our previous method was the inclusion in the reaction mixture of inorganic pyrophosphatase, which eliminates any competition from the reverse reaction and therefore improves the linearity of the rate plots, but does not affect the outcome of the experiment. For some proteins (wild-type, H734A, R754A, K758A, F762A, and Y766A), the steady-state kinetic parameters were determined in an identical manner for dGTP incorporation into a poly(dC)-oligo(dG) substrate.

Kinetic Analysis of Pyrophosphorolysis

The kinetic constants, K(m) and k, for the reverse reaction were determined using a DNA substrate made by annealing 5`-labeled oligo(dT)(14) to poly(dA) at a ratio of approximately 1 (dT)(14) primer per 300-500 nucleotides of poly(dA). The reaction mixture contained approximately 1 nM DNA in 50 mM Tris-HCl, pH 7.5, 16.5 mM MgCl(2), and varying pyrophosphate concentrations. For each enzyme, at least six PP(i) concentrations were used, chosen so as to bracket, if possible, the K(m) value of the Klenow fragment derivative. This was not possible for the R754A protein, which has a very low affinity for PP(i) (K(m) > 3 mM), since the reaction appears to be inhibited if the PP(i) concentration exceeds 2 mM, regardless of the Mg concentration. The pyrophosphorolysis reaction was initiated by adding wild-type or mutant Klenow fragment to a concentration of at least 340 nM; for each enzyme tested, a preliminary experiment established that this large excess of enzyme was adequate to bind all of the substrate, since the reaction rate was not changed by a further increase in enzyme concentration. Samples (4 µl) were removed at appropriate time intervals, during incubation at 20 °C, and quenched with 6 µl of 67% (v/v) deionized formamide containing 33 mM EDTA, 0.33% (w/v) sodium dodecyl sulfate, and 0.007% (w/v) each of bromphenol blue and xylene cyanol FF. The samples were fractionated on a denaturing 12% polyacrylamide gel and the relative amounts of the p(dT)(14) starting material and its degradation products were quantitated on a BAS 2000 Bio-Imaging Analyzer (Fuji) using the MacBAS Image Analysis software (Fuji). Reaction rates at each PP(i) concentration were calculated taking account of the fact that the reaction products are potential substrates(30) . For each time point, the mole fraction of each species was multiplied by the number of phosphodiester cleavages needed to generate that species (i.e. (fraction 13-mer) times 1 + (fraction 12-mer) times 2 + (fraction 11-mer) times 3 etc.), giving the total amount of pyrophosphorylytic degradation that had taken place at that time. (No correction was made for the small amount of polymerase-catalyzed extension that occurs as dTTP is formed by pyrophosphorolysis. The amount of dTTP produced is so small compared to the concentration of PP(i) in the reaction that interference from the forward reaction is insignificant, as indicated by the observation that the amount of p(dT) produced never exceeded 2% of the total counts.) The initial rates (V) at a series of PP(i) concentrations (S) were plotted using the Lineweaver-Burk formulation (double-reciprocal) to give K(m) and k.

Measurement of the Effect of Thiophosphoryl Substitution on the Polymerase Reaction

Single-turnover rates of incorporation of dTTP and dTTPalphaS by the R754A and K758A proteins were determined as described by Polesky et al. (5) with minor modifications. The DNA substrate was 5`-labeled (dT)(14) annealed to poly(dA), as described above for the pyrophosphorolysis reaction. The amount of the p(dT)(14) substrate remaining at each time point was normalized to a non-annealed 17-mer oligonucleotide, using the BAS 2000 scanner as described above. The reactions were carried out at 0 °C so as to slow the rate of reaction with the oxy substrate sufficiently that the rate could be measured using manual sampling. For the R754A mutant protein, the reaction rate was measured at 4 different nucleotide concentrations for each substrate (dTTP or dTTPalphaS) and the corresponding K(m)(dTTP) and K(m)(dTTPalphaS) and rate constants (k(o) and k(s)) were determined by extrapolation from a Lineweaver-Burk plot. For K758A, the elemental effect (k(o)/k(s)) was measured at 150 µM dTTP or dTTPalphaS (see Footnote 2).

Measurement of DNA Binding Affinity

The dissociation constant, K(d), for all the Klenow fragment derivatives in this study was determined using a gel mobility shift assay. The DNA substrate was the 68-mer hairpin oligonucleotide whose sequence is listed under ``Experimental Procedures''; this sequence forms a 26-base pair stem bounded by a stable tetraloop (31) and has a 12-nucleotide single-stranded 5` extension (see Fig. 4A). It was labeled at the 5` end using T(4) polynucleotide kinase and [-P]ATP. For a typical gel-shift assay, the labeled 68-mer was diluted to a concentration of 0.015 nM in 10 mM Tris-HCl, pH 7.5, 6 mM MgCl(2), 10% (v/v) glycerol, and 0.05% (v/v) Nonidet P-40. Klenow fragment (3 µl) was added to 12 µl of this DNA solution and the mixture was incubated at 4 °C for 10-15 min. A series of 10-15 concentrations of protein were used (see Fig. 4B), chosen so as to bracket the K(d) value, with the lowest protein concentration at least 3 times greater than the DNA concentration in the binding reaction. The DNA-protein samples were then loaded onto an 8% polyacrylamide gel (in 7.5 mM Tris borate, 2 mM MgCl(2), 0.1 mM EDTA) that had been pre-run for 1 h at 4 °C. The samples were loaded at 150 volts and then the voltage was reduced to 100 volts and the gel was run for 3-4 h at 4 °C. Because of the low ionic strength of the buffer it was necessary to circulate the reservoir buffers with a pump throughout the gel run and the buffer was changed after pre-running and before loading the samples. The gel was dried down, exposed to an imaging plate, and quantitated on the BAS 2000 scanner so as to determine the amount of complexed and uncomplexed 68-mer. The fraction of DNA bound was plotted against enzyme concentration and K(d) (the concentration at which 50% of the DNA is bound) was determined by interpolation.

Additionally, K(d) was determined for wild-type Klenow fragment and the R682A, H734A, R754A, K758A, and Y766A mutant derivatives using DNase I footprinting. The experiment was carried out as described previously(4) , except that the hairpin 68-mer was used instead of a primed M13 substrate, and the data were quantitated on a PhosphorImager instead of by densitometry of autoradiographs.


RESULTS

Site-directed Mutagenesis

The residues chosen for mutation (Fig. 1) are those which are close to or in contact with the bound dNTP in the crystalline binary complex of Klenow fragment with dCTP(20) . In each case the wild-type side chain was mutated to alanine. With the exception of Tyr, the mutated positions were residues that we had not previously investigated; in the case of Tyr, the new data for the Ala substitution complements the earlier studies on the Phe and Ser mutations(4, 32) . The mutations were made and the mutant Klenow fragment derivatives were overproduced and purified as described previously(4, 5) . All the Klenow fragment derivatives used in this study were deficient in 3`-5` exonuclease activity by virtue of the D424A mutation(17) . Since the polymerase activity of D424A is indistinguishable from that of wild-type Klenow fragment, and since this study focuses on the polymerase domain, we shall describe each protein in terms of the genotype of the polymerase region; thus the D424A control will be referred to as wild-type.

Melting Temperature

The aim of a mutational study of this type is to relate the observed differences between wild-type and mutant proteins to the specific amino acid substitutions that have been made. It is therefore important to rule out the possibility that a particular mutation may have caused changes in the three-dimensional structure of the protein that are not confined to the position of the altered side chain. Since such changes in structure could result in destabilization of the protein, we have measured the denaturation temperature (T(m)) for all the Klenow fragment derivatives in this study. We used circular dichroism spectroscopy to determine the alpha-helical content of each mutant protein as a function of temperature. The T(m) value obtained in this way for wild-type Klenow fragment (55 °C) is identical to the value derived from thermolysin digestion in our previous study(4) . The values for the mutant proteins (Table 1) were essentially identical to that of wild type, indicating that none of these mutations has caused a major distortion to the three-dimensional structure.

Kinetic Parameters for the Polymerase Reaction of Wild-type and Mutant Klenow Fragment Derivatives

The steady state parameters, K(m) and k, were determined for all the proteins in this study (Table 1). The use of a homopolymer DNA substrate, oligo(dT) annealed to poly(dA), simplifies the kinetic analysis since only one dNTP substrate is present, and allows us to compare our results with published values determined using the same substrate and reaction conditions(4, 5, 22) . Fig. 2(panels A and B) shows typical examples of the data obtained, the Eadie-Hofstee plots for the wild-type and R754A Klenow fragment derivatives (from which K(m) and k can be derived). The values determined for the ``wild-type'' control (which in fact carries the D424A mutation) were in good agreement with values previously determined for exonuclease-proficient wild-type(4, 5) ; it is unlikely that the 2-fold difference in k represents a significant difference in the behavior of the two proteins. The K(m) values for two of the Klenow fragment derivatives, R754A and F762A, were substantially higher than that of wild type (Table 1). Mutation at the intervening Lys position did not change K(m); however, this mutation caused the largest reduction in k of any of the mutations in the present study.


Figure 2: Eadie-Hofstee plots of the steady-state kinetic data for the polymerase reaction catalyzed by wild-type Klenow fragment (A), and the R754A (B), and H734A (C) mutant proteins. In each case, V represents the rate of incorporation of labeled dTTP, and S is the concentration of dTTP. Note that the axis scales are different in the three panels.



The H734A mutant protein was unusual in that the Eadie-Hofstee plot was not linear, but appeared instead to be composed of two linear plots (Fig. 2, panel C). Since this type of plot might be obtained if the mutant protein preparation were contaminated with a small amount of wild-type Klenow fragment, we repeated the determination with another preparation of the H734A protein from a different batch of induced cells. The same biphasic curve was obtained with the new enzyme preparation, indicating that this unusual behavior is indeed a property of the H734A mutant protein. Analysis of the rate data for H734A at relatively low concentrations of dTTP gave a K(m) of 10 µM; at dTTP concentrations above about 10 µM the measured rates were higher than expected based on a linear extrapolation, corresponding to an apparent activation by substrate dNTP. Substrate activation can be explained by the binding of a second molecule of substrate in such a way that the reaction rate is enhanced(33) . Analysis of the steeper portion of the Eadie-Hofstee plot (Fig. 2, panel C) indicated that the putative second binding site has a low affinity for dTTP (K(m) of around 300 µM) and causes the reaction rate to increase about 20-fold (values in parentheses in Table 1).

The steady-state kinetic constants for dGTP incorporation on a poly(dC) template were determined for wild-type Klenow fragment and those mutant proteins (H734A, R754A, K758A, F762A, and Y766A) that showed the largest differences from wild type in dTTP incorporation. We felt it was important to check the kinetics with a different dNTP substrate because of a recent study of the K758A mutant of Klenow fragment (34) that reported different kinetic constants for dGTP and dTTP incorporation. Our results showed interesting differences in the response of the mutant proteins to the two dNTP substrates (Table 1). For most of the proteins studied, K(m) was higher than K(m); the K758A mutant was the only one for which K(m) was significantly higher than K(m). For the H734A derivative the two K(m) values were approximately equal but the use of dGTP eliminated the apparent substrate activation seen with dTTP.

Kinetic Parameters for the Pyrophosphorolysis Reaction

Determinations of K(m) and k for the reverse reaction were carried out for wild-type Klenow fragment and for the R682A, H734A, R754A, K758A, and F762A mutant proteins. The R754A, K758A, and F762A proteins were chosen because they showed the largest changes in the kinetic constants for the forward reaction (see Table 1); mutations at Arg and His were also included because these side chains appear to interact with the dNTP beta- and -phosphates and/or with pyrophosphate in the respective binary complexes(20) . The experiment was carried out using a homopolymeric substrate (oligo(dT)(14) annealed to poly(dA)) with the enzyme in large excess over the DNA. Although the reaction was allowed to proceed beyond cleavage of the first phosphodiester bond, the rate remained linear (Fig. 3), suggesting that the rate-limiting step is at or before bond cleavage, in agreement with Dahlberg and Benkovic(8) . By measuring the rate at several different pyrophosphate concentrations, K(m) could be calculated (Fig. 3). The value we obtained for wild-type Klenow fragment is identical to that reported by Dahlberg and Benkovic(8) . Most of the mutant proteins tested had K(m) close to the wild-type value (Table 2). The exception was the R754A mutant which had a K(m) that was around 60-fold greater than that of wild-type Klenow fragment, suggesting that Arg interacts with the beta- or -phosphate of the dNTP.


Figure 3: Pyrophosphorolysis reaction catalyzed by the K758A mutant of Klenow fragment. Panel A shows the electrophoretic separation of aliquots of the reaction mixture removed at the time intervals (in hours) indicated at the top of each lane. The positions of the substrate, p(dT)(14), and the first three degradation products are indicated at the right-hand side. Panel B shows the rate plot from the experiment in Panel A; the number of phosphodiester bonds broken per molecule of starting material is plotted as a function of time.





Sulfur Elemental Effect

Since the rates of both the forward and reverse reactions are substantially reduced by the R754A and K758A mutations, a likely interpretation is that these mutations have caused the chemical step of the reaction to become rate-limiting. (^3)As we have previously shown, the observation of a sulfur elemental effect is diagnostic for a circumstance in which chemical catalysis is at least partially rate-limiting(5) . The elemental effect was determined for the R754A and K758A proteins by comparing the single turnover rate of incorporation of alpha-thio-dTTP with that for the normal oxy substrate. We made one important modification to our previous protocol because the mutations being studied were in side chains that were likely to be close to the dNTP alpha-phosphate, and we could not therefore assume that K(m) and K(m) would be equal, as they are for the wild-type protein(35) . For the K758A mutant protein, the elemental effect was measured at a high concentration of nucleotide (150 µM) to ensure saturation. The R754A mutant protein was more problematic because its high K(m) meant that it would be almost impossible to achieve a saturating concentration of nucleotide. Instead, rates were measured at several nucleotide concentrations and the maximal values of k(o) and k(s) were determined by extrapolation. This procedure also allowed estimation of K(m) and K(m) and indicated that for R754A K(m) is about 5-fold greater than K(m). The R754A protein gave a large elemental effect (k(o)/k(s) = 40), implying that the chemical step is rate-limiting. The smaller elemental effect (k(o)/k(s) = 5) observed for the K758A protein suggests that in this case the chemical step is only partially rate-limiting; given the large decrease in k caused by the K758A mutation, this would imply a substantial decrease in the rates of both the chemical step and the preceding conformational change.

DNA Binding Affinity

In the course of this work we developed a gel mobility shift assay for measurement of the dissociation equilibrium constant, K(d), for DNA binding to mutant derivatives of Klenow fragment. This method is quicker and easier than our previous DNase I footprinting method(4) , and is therefore better suited for scanning a series of mutant proteins for DNA binding defects. Using a small hairpin DNA oligonucleotide having a 26-base pair stem and a 12-nucleotide single-stranded 5` extension (Fig. 4A), the majority of the labeled material enters the gel and two major bound species can be seen (Fig. 4B). We believe that the faster running complex (B, in Fig. 4B) is the expected species having one molecule of polymerase bound at the DNA 3` terminus, while the slower complex (A, in Fig. 4B), which appears at higher protein concentrations, contains one or more additional protein molecules. The binding conditions used, low temperature and low ionic strength buffer, were found to be ideal for minimizing the smearing of the protein-DNA complex bands, especially for those mutant proteins which have a lower affinity for DNA. For quantitation purposes, the bound DNA fraction was considered to comprise labeled material in both discrete complex bands as well as the radioactive material that formed a faint smear between the complex bands and the position of unbound DNA (the latter presumably corresponds to complexes that have dissociated during the running of the gel). For each determination, data from 10-15 protein concentrations were plotted to give a binding curve from which K(d) was calculated (Fig. 4C). The results (Table 3) showed that the R682A, H734A, and Y766A Klenow fragment derivatives have substantially reduced DNA binding affinity, suggesting that the corresponding residues could be making contact with the DNA substrate.



As a check on the validity of the gel mobility shift method, K(d) was also determined by DNase I footprinting for the wild-type, R682A, H734A, R754A, K758A, and Y766A proteins. The results are in good agreement with K(d) values determined from the gel assay (Table 3), and give us confidence that gel mobility shifts give a reliable estimate of K(d) in this system.


DISCUSSION

Mutations That Affect dNTP Affinity

To identify those amino acid residues that are responsible for making contact with the incoming dNTP molecule in the catalytically competent ternary complex, we have made mutations at seven positions (Arg, Gln, His, Arg, Lys, Phe, and Tyr) all of which are close to or in contact with the dNTP in the Klenow fragment-dNTP binary complex(20) . All the residues mutated in this study are invariant or highly conserved residues in the pol I family of DNA polymerases (Fig. 5; (10) and (36) ), suggesting that they play an important role in the polymerase reaction. Measurement of the denaturation temperature of the mutant proteins indicates that none of the mutations causes a gross alteration of the protein structure. Moreover, since the mutated side chains are all on the surface of the protein (see Fig. 1), it is probably reasonable to assume that the structural changes resulting from each mutation are confined to the position of the mutated side chain. This assumption is necessary in order to draw meaningful conclusions from a study of this type.


Figure 5: Conservation of the residues mutated in this study in the pol I (or A) family of DNA polymerases. The alignment of the relevant regions of E. coli DNA polymerase I with the DNA polymerases from Streptococcus pneumoniae (Sp), Thermus aquaticus (Tq), Thermus flavus (Tf), bacteriophages T5, T7, SP01, and SP02, and the yeast mitochondrial DNA polymerase (Mip1) is based on that of Braithwaite and Ito(36) . To this alignment have been added the sequences for the DNA polymerases from Thermus thermophilus (Tt)(45) , Deinococcus radiodurans (Dr)(46) , Bacillus caldotenax (Bc)(47) , Mycobacterium tuberculosis (Mtb) (V. Mizrahi, personal communication (GenBank accession no. L11920)), Mycobacterium leprae (Mlep) (D. R. Smith, personal communication (GenBank accession no. U00021)), bacteriophage T3(48) , and mycobacteriophage L5(49) . The residues relevant to this study (which are by no means the only conserved residues within the regions shown) are boxed; dark grey shading indicates identity with the E. coli prototype sequence, and lighter grey indicates a conservative substitution.



To measure dNTP binding, we first determined K(m) values for the polymerase reaction for each of the mutant Klenow fragment derivatives, since K(m) has previously been shown to be a valid measure of the binding affinity of dNTP (K(m) = K(d)) in the catalytically competent ternary complex(6, 22, 37) . Substitution of Arg and Phe caused K(m) to increase by 2 orders of magnitude. This is by far the largest increase seen for any Klenow fragment mutation reported to date, and translates into a loss of about 3 kcal mol in binding energy. (Aside from the present study, 23 single amino acid mutations comprising substitutions at 13 different positions in the polymerase domain of Klenow fragment have been studied in detail(4, 5, 32, 34, 37, 38) ). Although measurements of K(m) for a mutant protein cannot distinguish between direct effects (due to removal of a side chain that contacts the dNTP molecule) and indirect effects (via a protein contact to the template strand), we suspect that the large increases due to the R754A and F762A mutations reflect direct contacts of Arg and Phe with the incoming dNTP. Conversely, the smaller changes in K(m) that resulted from the Y766A and H734A mutations are suggestive of indirect effects via template strand contacts, a conclusion that is supported by the observation that both mutations caused a decrease in DNA binding affinity. The mutations R682A and Q708A, in residues that contact the dNTP in the crystalline binary complex(20) , also had extremely modest effects on dNTP binding, arguing against direct contacts in the ternary complex.

Interactions with the negatively charged phosphate groups of the dNTP molecule are likely to account for an important subset of the Klenow fragment-dNTP interactions. Certainly, earlier studies on the binary complex indicated that the dNTP phosphates are important determinants of binding specificity(39) . Moreover, pyrophosphate is a competitive inhibitor of the polymerase reaction, suggesting that the PP(i) binding site overlaps the dNTP binding site in the ternary complex(8) . Comparison of the values of K(m) and K(m) (Fig. 6) suggests that the positively charged Arg interacts with the beta- and/or -phosphates of the dNTP, whereas Phe contacts some other part of the molecule. Given the hydrophobic nature of the Phe side chain, a plausible contact would be with the deoxyribose, or with that portion of the base not involved in contacts with the opposing template strand.


Figure 6: Relative K(dTTP) (A) and K(PPi) (B) values for wild-type and mutant Klenow fragment derivatives. Values are normalized to the wild-type value, set at 1.



The comparison between K(m) and K(m) for selected mutant proteins showed some interesting differences in the utilization of these two substrates. Wild-type Klenow fragment bound dGTP 4-fold more tightly than dTTP in the ternary complex (as measured by the ratio of the two K(m) values), perhaps reflecting the stronger base pairing of dGTP with the template. Although the K(m) values were much higher, the R754A mutant protein gave the same ratio, indicating that the binding of both dGTP and dTTP is impaired to a similar extent, as would be expected for a mutation that affects a phosphate contact remote from the nucleotide base. By contrast, the other mutant proteins tested gave quite different ratios, ranging from K758A, which affected dGTP binding more than dTTP binding, to F762A for which the opposite was observed. Thus it appears that, in the region where the protein contacts the dNTP base and ribose, the side chains assume different degrees of importance depending on the exact circumstances. With the homopolymer DNA substrates used in our assays, Phe is the most important contact (in addition to the phosphate contact at Arg) when T is the incoming base, whereas Lys and Phe contribute approximately equally when G is added. Mutation of His also has a substantial effect on dGTP binding; however, as indicated above, we suspect that this effect may be mediated via the DNA template. Presumably the apparent changes in protein contacts for different incoming dNTPs reflect subtle differences in geometry in the respective ternary complexes; however, our data do not allow us to distinguish between several possibilities that may form the basis for these differences. The polymerase could have a distinct mode of interaction with each of the four bases; alternatively, it might distinguish between purines and pyrimidines, or between A-T versus G-C pairings. A more complex but perfectly plausible scenario is that the geometry of the dNTP interaction is influenced by the local sequence context at the position of insertion. This latter possibility is certainly consistent with the report by Eger et al.(37) of two distinct K(d)(dTTP) values for wild-type Klenow fragment on non-homopolymeric substrates, both of which were substantially higher than the K(m)(dTTP) reported here for incorporation on poly(dA)-oligo(dT).

We do not have an explanation for the apparent substrate activation shown by the H734A mutant protein at high dTTP concentrations. This type of kinetic behavior is relatively rare (e.g. refs. 33, 40); when observed it has been attributed to a second, weaker substrate binding site whose occupancy leads to an increase in reaction rate. The reaction rates have been measured for wild-type Klenow fragment and for each of the mutant derivatives in this study over the same range of dTTP concentration as in the H734A determination, but none of the proteins (aside from H734A) showed any unusual relationship between reaction rate and dTTP concentration. It is unclear at present why the H734A mutation, which probably removes a DNA contact, should allow this aberrant binding of a second dTTP molecule, or why the effect should be seen with dTTP but not with dGTP.

Mutations That Affect DNA Binding and Catalysis

Current models for the binding of DNA to Klenow fragment place the primer terminus and the uncopied template strand beyond the site of synthesis in the large cleft on the polymerase domain, with the template strand likely to make contact with residues on the exposed face of Helix O (Fig. 1)(27) . Of the residues involved in the present study, all of which are located in the proposed DNA contact region, the largest contributions to DNA binding are made by Arg, His, and Tyr. Model building suggests that the template strand passes close to Tyr(27) , and our own preliminary data indicate that His interacts with the single-stranded template beyond the site of nucleotide addition. (^4)Mutations in Arg, Lys, and Phe, had little or no effect on DNA binding, supporting our suggestion that these side chains are important in making direct (not template-mediated) contacts with the incoming dNTP.

In addition to their effect on K(m), the R754A and K758A mutations caused a substantial decrease in the rate of the polymerase reaction. The sulfur elemental effect data imply that loss of Arg slows the chemical step of the reaction, while Lys may be involved to some extent in both the chemical step and the preceding conformational change. The roles of the Arg and Lys side chains in accelerating the polymerase reaction could involve stabilization of the relevant transition state(s) via interactions that are related to those proposed with the dNTP ground state. For Arg, we suggest that the interaction with the beta- or -phosphate may assist in the removal of the pyrophosphate leaving group as the chemical step of catalysis proceeds. For Lys, it seems that the extent to which the interaction between this side chain and the dNTP molecule is manifested in the ground state or in the relevant transition state(s) varies depending on the identity of the incoming dNTP. When dTTP is the incoming nucleotide, Lys stabilizes the transition state without contributing to ground state binding, since k is decreased 300-fold while K(m) is virtually unchanged. Given the positive charge on the Lys side chain, it is possible that it may interact so as to neutralize the developing negative charge at the alpha-phosphate oxygens. Whatever the nature of the interaction that takes place at Lys, it is clear that, when dGTP is the incoming nucleotide, more of the binding energy is used in the ground state and consequently less is available for transition state stabilization. This is shown by the observation that the K758A mutation has a greater effect on K(m) and a smaller effect on k when addition of dGTP is compared with addition of dTTP.

Relationship to Previous Mutational Studies of Klenow Fragment

This study, together with our previous studies of Klenow fragment mutants(4, 5) , has identified an area of the polymerase cleft that is implicated in making contacts, directly or indirectly, with dNTP in the ternary complex. This area is on the side of the cleft that forms the fingers subdomain and encompasses the N-terminal portion of Helix Q (Arg and Asn), one face of Helix O (Arg, Lys, Phe, and Tyr), and neighboring residues between Helix O and the base of the cleft (Asp, Glu, and His). Since these side chains cover an area that is too large to be spanned by a single dNTP molecule, it is clear that not all of these residues can be in direct contact with the dNTP. The data reported here suggest that the region including Arg, Lys, and Phe provides direct contacts to the dNTP molecule (the precise details of these contacts varying to some extent with the nature of the incoming dNTP). Mutations of side chains outside of this region tend to have smaller effects on K(m), as shown in Table 1and in our previous results for mutations at Asp, Glu, Tyr, Arg, and Asn, in which the largest K(m) was about 8-fold higher than the wild-type value(4, 5) . These smaller effects are more compatible with a model in which the mutated side chains do not contact the dNTP molecule directly; as noted above, several of these residues may influence dNTP binding via an interaction with the DNA template, and others (Asp and perhaps Glu) are involved in coordinating the divalent metal ions at the polymerase active site. (^5)

Our data for the Y766A mutation can be compared with previous studies on the Y766S and Y766F mutations (4, 32) to give a fuller picture of the requirements at this position. The Ala substitution is slightly more detrimental than the Ser substitution, while the Y766F protein behaves very similarly to wild-type Klenow fragment (32) . (^6)Like Y766S, but unlike Y766F, the Y766A mutation has a mutator phenotype in vivo (data not shown). Thus, the phenolic ring appears to be the most important feature of the Tyr side chain, with the hydroxyl group playing a significant but secondary role, consistent with the observation that Tyr (not Phe) is invariably found at this position in related polymerase sequences (Fig. 5; (10) and (36) ).

Two of the mutations in the present study, R682A and K758A, have also been investigated recently by Pandey et al.(34, 38) , and our results are largely in agreement with theirs although we differ on some points of interpretation. For the K758A mutant protein, Pandey et al.(34) reported essentially identical kinetic constants to ours, using similar homopolymer substrates, and likewise noted a difference in the incorporation of dTTP and dGTP. However, we disagree with their suggestion that Lys may interact with the dNTP beta- or -phosphate or that it plays a specific role in translocation. First, our observation that the K758A mutation did not affect pyrophosphate binding argues against an interaction with the beta- or -phosphate. Second, the K758A protein showed a slow rate of reaction under the single turnover conditions employed in our elemental effect measurements, indicating a slow step at or preceding chemistry (see also Footnote 3). Since processivity is determined simply by the competition between the forward reaction and DNA dissociation, it is not necessary to postulate, in addition, a slowing of translocation in order to account for the lower processivity observed by Pandey et al.(34) , although the data do not of course rule out another slow step at a later stage in the reaction pathway. In contrast with the results of Pandey et al.(34) , we have not observed any particular tendency of the K758A protein to pause at template A residues. (^7)For the R682A protein Pandey et al.(38) reported a small increase in K(m) and a 20-25-fold decrease in k, whereas we have found the values to be almost the same as wild type. A possible explanation for this disagreement is that the homopolymer substrate used by Pandey et al.(38) in their experiments apparently contained a DNA primer annealed to an RNA template, whereas our assays employed the usual DNA-DNA hybrid. As with the K758A mutation, we do not feel that the data for R682A support a specific role for this side chain in translocation, as suggested by Pandey et al.(38) , since the observed decrease in processivity could be simply a consequence of weaker DNA binding. Instead, we suggest that the rather insignificant effects of the R682A mutation on the polymerase reaction are probably related to the decrease in DNA binding affinity caused by this mutation. The lack of any dramatic effect of the R682A mutation on dNTP binding was somewhat surprising, given the results of affinity labeling experiments in which this side chain was labeled by 5`-fluorosulfonylbenzoyl deoxyadenosine in a template-dependent manner (25) . It is possible that the Arg side chain, while not participating in a specific interaction with the dNTP, may yet be sufficiently close to the reactive portion of the affinity analog to become labeled, especially since the reagent is selective for nucleophilic side chains.

Relationship to Other Polymerases

As shown in Fig. 5, the majority of the residues involved in the present study are invariant in the pol I (or A) family of DNA polymerases, and the remainder are highly conserved. The least well conserved residue, Gln, which is invariant only in the subgroup of nine bacterial polymerases and is not well conserved in the bacteriophage or mitochondrial polymerases, is also the residue whose replacement had the least effect on the polymerase reaction as judged by our assays. Although Phe, which appears to play an important role in dNTP binding, is not an invariant residue, its position is always occupied by a large hydrophobic side chain (Phe or Tyr in all but one instance), and this may be the important attribute at this position. All of the amino acid residues under discussion are embedded within conserved sequence motifs, two of which, labeled as the Gln region and Helix O in Fig. 5, correspond to the sequence motifs A and B of Delarue et al.(10) which appear to be conserved between more distantly related families of polymerases. Several mutations in the corresponding motifs of human DNA polymerase alpha and 29 DNA polymerase (also a member of the pol alpha family) have been shown to affect dNTP binding(41, 42, 43) , implying that these conserved sequence motifs may serve similar functions in the two different polymerase families. However, in the absence of a structure for any member of the pol alpha family, these apparent analogies should be viewed with caution. The recent structural data for mammalian DNA polymerase beta show that it may be misleading to use these rather tenuous sequence alignments to draw parallels between non-homologous polymerases(13, 14, 44) .

The Proposed dNTP Binding Site in the Ternary Complex

An important question that existed before the start of this work is the relationship between the catalytically competent ternary complex and the crystalline binary complex of Klenow fragment with dNTP. The results presented here are consistent with the prediction (20) that the triphosphate positions in the binary complex may be more relevant to the ternary complex than are the base and ribose positions. Of the residues seen to be close to the triphosphate in the binary complex, Arg appears to play an important role in the ternary complex, whereas Gln, Arg, and His make little or no contribution (the latter 2 residues may be involved instead in interactions with DNA in the ternary complex). Our data suggest that an interaction between Lys and the phosphate positions, which appears plausible from the binary complex structure, is felt to a greater extent in one of the transition states than in the ground state. Placing the phosphates near to Arg and Lys necessarily brings the deoxyribose close to Phe, and this is supported by our K(m) measurements. As pointed out by Beese et al.(20) , the position of the nucleotide base may well not be fixed in a catalytically appropriate position in the absence of a DNA template.

Data from a number of sources (reviewed in (9) ) indicate that the active site of DNA polymerases is defined by a trio of carboxylate groups (Asp, Asp, and Glu in Klenow fragment) which are proposed to anchor a pair of divalent metal ions that carry out catalysis of the phosphoryl transfer reaction (9, 18) . The results of this and our earlier studies (4, 5) allow us to make predictions about some of the other interactions that take place in the active site. The proposed reaction mechanism requires the dNTP to bind with its alpha-phosphate group positioned between the metal ions, and this expectation has been confirmed by structural studies of mammalian DNA polymerase beta(13, 19) . Our previous elemental effect data (5) suggest that the Sp oxygen at the alpha-phosphate is close enough to the Asp side chain to provide steric obstruction in the transition state. (^8)From the studies presented here, we propose also that the dNTP beta- and/or -phosphate interacts with Arg, and the uncharged portion of the molecule, perhaps the deoxyribose ring, is bound close to Phe. As the phosphoryl transfer reaction proceeds, polar side chains in the active site region (including Arg, Lys, Arg, and Gln, (5) ) may make interactions that stabilize the transition state and thus facilitate the reaction. Our current data suggest that Arg acts to assist in removal of the pyrophosphate leaving group; we do not have evidence to assign precise roles to the other three side chains at present. We hope that, in the future, these predictions can be tested, and the interactions at the active site defined in more detail, by crystallographic data for a ternary complex of Klenow fragment with DNA and dNTP substrates.


FOOTNOTES

*
This work was supported by Grant GM-28550 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, Bass Center for Molecular and Structural Biology, 266 Whitney Ave. (P. O. Box 208114), New Haven, CT 06520-8114. Tel.: 203-432-8992; Fax: 203-432-9782.

(^1)
The abbreviations used are: dNTP, deoxynucleoside triphosphate; dTTPalphaS, deoxythymidine 5`-O-(1-thiotriphosphate); PP(i), pyrophosphate; protein mutations are abbreviated using the following convention: the residue number is preceded by the symbol (in the one-letter code) for the wild-type amino acid and followed by the symbol for the mutant amino acid. Thus, R754A denotes a mutation from Arg to Ala at position 754.

(^2)
For calculation purposes, the concentration of the Sp diastereomer of dTTPalphaS was assumed to be half of the concentration of the mixture of diastereomers. Concentrations given in the text refer to the Sp diastereomer only; thus, for an experiment in which the concentration of the nucleotide substrate was 50 µM, the reaction in fact contained 100 µM of the mixed diastereomers.

(^3)
For both the R754A and K758A proteins, a primer extension reaction carried out on poly(dA)-oligo(dT) under conditions of enzyme excess indicated that the rates of the first two nucleotide additions were approximately equal. This observation implies a rate-limiting step at or before the chemical step of catalysis; a rate-limiting step later in the reaction pathway would result in the second addition being much slower than the first.

(^4)
C. M. Joyce, manuscript in preparation.

(^5)
L. S. Beese and T. A. Steitz, unpublished observations.

(^6)
C. M. Joyce, unpublished observations.

(^7)
C. M. Joyce, unpublished data.

(^8)
The previous elemental effect studies left open the possibility that Asp (the other catalytically important carboxylate of Klenow fragment) may likewise be close to the dNTP alpha-phosphate. Subsequent studies have shown that, not only do substitutions at Asp of the smaller side chains Ala and Ser give no sulfur elemental effect, but the same is true for the Asn and Glu substitutions. This result suggests that the side chain of Asp is not as close to the Sp oxygen position at the alpha-phosphate as is Asp.


ACKNOWLEDGEMENTS

We are grateful to Tom Steitz and colleagues, especially Joe Jäger, for their insights into the Klenow fragment structure. We also thank Xiaojun Chen Sun and Ruoying Tang for expert technical assistance, Joe Jäger for making Fig. 1, Joe Coleman for his insights on substrate activation, and Paul Predki and Lynne Regan for help with the circular dichroism measurements.


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