©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Studies of Human DNA Polymerase
LYSINE 950 IN THE THIRD MOST CONSERVED REGION OF alpha-LIKE DNA POLYMERASES IS INVOLVED IN BINDING THE DEOXYNUCLEOSIDE TRIPHOSPHATE (*)

(Received for publication, May 11, 1995; and in revised form, June 22, 1995)

Qun Dong Teresa S.-F. Wang (§)

From the Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The function of a lysine residue, Lys, of human DNA polymerase alpha located in the third most conserved region and conserved in all of the alpha-like polymerases was analyzed by site-directed mutagenesis. Lys was mutagenized to Arg, Ala, or Asn. The mutant enzymes were expressed in insect cells infected with recombinant baculoviruses and purified to near homogeneity. The mutant enzymes had specific activities ranging from 8 to 22% of the wild type. All three Lys mutants utilized Mn as metal activator more effectively than the wild type enzyme and showed an increase in Kvalues for deoxynucleoside triphosphate but not k values in reactions with either Mg or Mn as the metal activator. Although mutation of the Lys residue caused an increase in K values for deoxynucleoside triphosphates, mutations of Lys to Arg, Ala, or Asn did not alter the mutant enzymes' misinsertion efficiency in reactions with Mg as a metal activator as compared with that of the wild type, suggesting that the base of the incoming deoxynucleoside triphosphate is not the structural feature interacting with the Lys side chain. In reaction with Mn as a metal activator, all three Lys mutants had an improved fidelity for deoxynucleotide misinsertion compared to wild type. Inhibition studies of the three Lys mutant derivatives with an inhibitor, structural analogs of deoxynucleoside triphosphate, and pyrophosphate suggest that the deoxyribose sugar and beta-,-phosphate groups are not the structural feature recognized by the Lys side chain. Comparison of the mutant enzymes to the wild type enzyme for their affinities for dCTPalphaS versus deoxynucleoside triphosphate suggests that this highly conserved Lys is involved in interacting either directly or indirectly with the oxygen moiety of the alpha-phosphate of the incoming deoxynucleoside triphosphate.


INTRODUCTION

Compilation and alignment of the protein sequences of DNA polymerases deduced from the nucleotide sequence data have classified DNA polymerases into three families, family A, B, and C, according to their similarities to Escherichia coli polymerase I, II, and III, respectively(1, 2) . The alpha-like DNA polymerases including the E. coli polII belongs to family B withpolymerase alpha as the prototype(3) . The lack of an intrinsic proofreading nuclease in DNA polymerase alpha makes this enzyme an ideal model to study the structure-function relationship of the active site and serves as a model for all of the alpha-like DNA polymerases, particularly in identifying residues in the active site that are responsible for DNA synthetic fidelity(4, 5) . We have overproduced functionally active recombinant human DNA polymerase alpha catalytic subunit in insect cells infected with a recombinant baculovirus (6) and established a one-step immunoaffinity purification protocol to purify the enzyme to near homogeneity(7) . By site-directed mutagenesis followed by physical and steady-state kinetic studies, several highly invariant residues in the catalytic site of human DNA polymerase alpha were analyzed(4, 5, 8, 9) . We have established that three residues, -Asp-Thr-Asp-, in the most conserved region of the alpha-like DNA polymerases (region I, -YGDTDS-) are involved in metal activator binding. The two aspartate residues, Asp and Asp, like the aspartate residues in the active site of Klenow and HIV reverse transcriptase forming the ``catalytic triad,'' directly participate in chelating the metal ion(8, 9, 10, 11, 12) . Mutations of Asp to Asn and Thr to Ser also yielded mutant polymerases as metal ion-induced anti-mutators (8) . Thus, residues Asp, Thr, and Asp in the most conserved region also play critical roles in the observed metal-induced infidelity in DNA synthesis of cellular DNA polymerases.

Five highly conserved residues in the second most conserved region (region II) were also analyzed. Mutation of a conserved glycine, Gly, appears to affect both catalysis and substrate dNTP binding, suggesting that this glycine residue is essential for the maintenance of the overall active site structure. Mutations of Tyr altered the affinity of the mutant enzyme to bind the incoming dNTP. Analysis of the Tyr mutant enzyme for its DNA synthetic fidelity has shown that the phenyl ring side chain of Tyr directly interacts with the nucleoside base moiety of the incoming dNTP and plays a critical role in nucleotide misinsertion fidelity of DNA synthesis(5) . Mutation analyses of the second serine residues in the conserved region, -SLYPSI-, have revealed that the hydroxyl side chain of this serine residue, Ser, directly interacts with the 3`-OH terminus of the primer and plays an essential role in mispaired primer extension fidelity of DNA synthesis(4) .

In this report, we continue to investigate the contributions of those highly conserved amino acid residues in the catalytic site of alpha-like DNA polymerases for either substrate binding or for catalysis. We analyzed an invariant lysine residue in the third most conserved region (region III) that is conserved from human DNA polymerases alpha, , and to E. coli polymerase II.


EXPERIMENTAL PROCEDURES

Materials

Ultrapure deoxyribonucleotides and poly(dA) and oligo(dT) were purchased from Pharmacia LKB Biotechnology Inc. ddCTP and dCTPalphaS (^1)in S(p) diastereomer form and the [alpha-S]dCTPalphaS were from Amersham Corp. Aphidicolin and phosphonoacetic acid were obtained from Sigma. Butylphenyl-dGTP (BuPdGTP), butylphenyl-dGMPCH(2)PP (BuPdGMPCH(2)PP), carbonyldiphosphonate were gifts from G. Wright (University of Massachusetts, Boston). All other chemicals were from commercial sources and were analytical grade. Oligonucleotides for mutagenesis (5`-GCTGTGAGCCTCAAAGCCT-3`, 5`-TTCGCTGTGAGCGCCAAAGCCTTCTG-3`, and 5`-CGCTGTGAGGTTCAAAGCC-3`) were synthesized from an Applied Biosystems DNA Synthesizer.

Methods

Site-directed Mutagenesis

The cloning and in vitro mutagenesis strategy was as described(9) . Briefly, a 1.44-kilobase SalI-BamHI fragment of the human polymerase alpha cDNA containing the conserved regions (13) was cloned into the SalI-BamHI site of M13mp19 for site-directed mutagenesis in vitro(9) . The mutations were verified by restriction enzyme analysis and DNA sequencing.

Construction of Recombinant Baculovirus

The strategy for construction of transfer vector was performed as described in (9) . Briefly, the SalI-BamHI fragments containing the site-directed mutation were isolated and used to replace the SalI-BamHI fragment of the plasmid pBR(XbaI) HDPalpha which contains the full-length coding sequence of human DNA polymerase alpha(9) . A 4.6-kilobase XbaI-XbaI fragment of pBR(XbaI)HDPalpha containing the full-length mutated human polymerase alpha cDNA was then isolated and constructed into the XbaI site of pVL1393, to generate the transfer vector pVL1393/SDM containing the site-directed mutation in the polymerase alpha cDNA.

Recombinant baculoviruses were generated by co-transfection of Sf9 cells with the transfer plasmids and linear baculovirus DNAs as described(9) . Five micrograms of transfer plasmid DNA were co-transfected with 1 µg of linear AcbetaGal viral DNA, and the recombinant viruses were selected using standard baculovirus techniques.

Expression and Purification of Mutants

The amplification, infection, and harvest of recombinant baculoviruses expressing the mutant polymerase alpha were performed as described(9) . The recombinant DNA polymerase alpha proteins were purified to near homogeneity from Sf9 insect cell lysates by the one-step immunopurification protocol with monoclonal antibody SJK237-71 cross-linked to Sepharose 4B as described(7) .

DNA Polymerase Assay and Kinetic Analysis

The standard assay for DNA polymerase alpha activity using optimally gapped calf thymus DNA as primer-template was as described(14) . One unit of polymerase activity is defined as the amount of DNA polymerase that incorporates 1 nmol of labeled dNTP into acid-insoluble DNA at 37 °C in 1 h with 10 mM MgCl(2). All kinetic assays and metal ion optimum curves were performed with optimally gapped calf thymus DNA which was extensively dialyzed to remove the Mg ion as described(9) . The metal optimum curves, k and K(m), were determined using optimally gapped calf thymus DNA as the primer-template substrate. K(m) for primer-template was determined by varying the concentrations of oligo(dT)/poly(dA) as described(6) . Initial rate kinetic data were analyzed by using the computer program CRICKET GRAPH.

Processivity

Processivity assays for wild type and mutant enzyme were performed by using the DNA trap method described in (15) . Oligo(dT) primer was 5`-P-end-labeled and annealed 1:1 to (dA) in 20 mM KCl, 10 mM Tris HCl, pH 8.0. The labeled primer-template (0.45 pmol) was mixed with 0.13-2.8 pmol of enzyme in a volume of 5 µl containing 25 mM Tris-HCl, pH 7.5, 0.1 mg/ml acetylated bovine serum albumin, and 5 mM MgCl(2) or 1 mM MnCl(2). The synthesis reaction was initiated by the addition of 5 µl of 100 µM dTTP for 20 s at room temperature and then quenched by the DNA trap (10 mg/ml activated calf thymus DNA, 60 µM dTTP, 20 µM dCTP, 20 µM dATP, and 20 µM dGTP) at 37 °C for 2-10 min depending on the activity of the mutant. Control reactions were carried out by the addition of 5 µl of 100 µM dTTP without the DNA trap.

Misinsertion Fidelity

The fidelity of misinsertion by wild type and mutant polymerase alpha was measured by the gel electrophoresis assay described in (5) and (16) -18. The standing start primer-template used to measure correct or incorrect insertion was: 5`-TGA CCA TGT AAC AGA GAG-3` (18-mer) and 3`- ACT GGT ACA TTG TCT CTC ATT CTC TCT CTC TTC TCT-5` (36-mer), where the bold and underlined nucleotide on the template indicates the position of insertion of either the correct dTMP or the incorrect dCMP. The oligonucleotides were gel-purified before use. The primer was 5`-end-labeled with P and annealed 1:2 to the template. The primer-template (0.5 pmol) was mixed with 0.3 unit of the enzyme in a 10-µl reaction buffer containing 20 mM Tris-HCl, pH 8.0, 1 mM beta-mercaptoethanol, 200 µg/ml acetylated bovine serum albumin, and either with 10 mM MgCl(2) or with 0.75 mM MnCl(2) as metal activator. The reaction was initiated by the addition of either dTTP (correct) or dCTP (incorrect) to fill the I(1) position in the primer for 2 min at 37 °C and terminated on a dry ice bath followed by the addition of an equal volume of 95% formamide sequencing gel loading dye. Reaction products were boiled for 5 min and separated on a 13% polyacrylamide sequencing gel. After electrophoresis, gels were analyzed by PhosphorImager (Molecular Dynamics). I(0) designates the primer band, and I(1) designates the site of correct or incorrect insertion. Velocity was measured as I(1)/(I(0) +I(1)), and K(m) and V(max) values were deduced from Lineweaver-Burk plots.

Single-stranded DNA Inhibition

Wild type or mutant enzyme (1.4 pmol) was preincubated with varying concentrations of (dT) for 5 min at 0 °C in a 10-µl incubation and then assayed under standard conditions with optimally gapped DNA as primer-template.

Inhibition Assay

Enzymatic assays with (40 to 400 pmol) of wild type or mutant polymerase alpha enzymes were carried out in the presence and absence of inhibitors in a reaction mixture containing 20 mM HEPES (pH 8.0), 2 mM beta-mercaptoethanol, 200 µg/ml bovine serum albumin, 10 mM MgCl(2), 50 µM dNTP, and 800 µg/ml gapped calf thymus DNA at 37 °C for 10 min. The concentration of inhibitors that produced 50% inhibition (IC) are mean values from two or three independent assays.


RESULTS

Site-directed Mutation of a Highly Conserved Lysine Residue

A lysine residue located in the third most conserved regions of the alpha-like DNA polymerases is invariantly present in all three major mammalian cellular DNA polymerases alpha, , and , in yeast POLI, -II, and -III, in several DNA virus polymerases, in E. coli polII, in polymerases of bacteriophage T4, PRD1, and 29, and in polymerase-like proteins such as PGKL1 and mitochondria S1 (Fig. 1). In this study, we used human DNA polymerase alpha as the model for all of the three cellular polymerases to analyze the functional role of this highly conserved lysine residue. By site-directed mutation, we changed Lys to Arg, thereby replacing the side chain with a larger positively charged side chain, Lys to Ala, thereby completely abolishing the positively charged side chain, and Lys to Asn, thereby replacing the -NH(3) group of the positively charged lysine side chain with a polar amide group. The three mutant polymerases were produced in recombinant baculovirus-infected insect cells and purified with the one-step immunopurification protocol to near homogeneity in high yield(7) . Analysis of these three mutant proteins produced from recombinant baculovirus-infected insect cells showed that all three had a predominant protein of 180 kDa like the wild type enzyme with minor species of 165-140 kDa (data not shown). Furthermore, these three mutant proteins like the mutant proteins reported by us before had no detectable global structural alterations(4, 5, 9) . The specific activities of each mutant DNA polymerase alpha were measured by using optimally gapped calf thymus DNA as primer-template and with either Mg or Mn as metal activator. In reactions with Mg as the metal activator, mutant K950A had 22% of the specific activity of the wild type enzyme, while mutants K950R and K950N had 7.6% and 8% of the wild type specific activity, respectively. In reactions with Mn as metal ion, mutant K950R had 42% of the wild type specific activity, mutant K950A had 70% of the wild type specific activity, while mutant K950N had specific activity identical with, if not slightly higher than, the wild type enzyme (Table 1).


Figure 1: Sequence alignment of the third most conserved region of alpha-like DNA polymerases. Amino acid residues 943-967 of human DNA polymerase alpha were aligned with amino acid residues from 18 other alpha-like DNA polymerases. Gaps are indicated by dashes, and extensive gaps are indicated by the number of amino acids contained within the gap. All of the amino acid sequences are derived from (1) and (2) . The amino acid sequences of human polymerase , bovine polymerase , E. coli DNA polII, Schizosaccharomyces pombe polymerases alpha, and are from (27, 28, 29, 30) , respectively. Conserved residues are boxed. The highly conserved lysine residue studied in this report is marked by number sign. Two other highly conserved residues, tyrosine (Y) and glycine (G), are marked with *.





Kinetic Parameters of the LysMutants

Kinetic parameters of these three mutants in reactions with either Mg or Mn as the metal activator were measured and compared to the wild type (Table 1). We found that mutations of Lys to Arg, Ala, or Asn had a profound effect on the K(m) values of dNTPs in reactions with Mg as the metal activator. Mutants K950R, K950A, and K950N showed 76-, 25-, and 42-fold increases of K(m) for dNTP, respectively, as compared to the wild type. These mutants, however, only showed moderate 2- to 5-fold decreases in their k values in reactions with Mg. Moreover, in reactions with Mg, all three Lys mutant derivatives showed striking decreases in their processivity to the nearly distributive mode of DNA synthesis. All of the three Lys mutant enzymes also showed moderate decreases in their K(m) values for primer terminus compared to the wild type enzyme in reaction with Mg. These results suggest that in reactions with Mg as the metal activator, the observed lower specific activities of the Lys mutant derivatives as compared with the wild type are due to the combination of increases in the K(m) for dNTP and decreases in k and processivity.

In reactions with Mn as the metal activator, mutants K950R, K950A, and K950N had 8-, 4-, and 6-fold increases in K(m) for dNTPs and moderate 3-, 3.5-, and 4.5-fold increases in their k, as compared with the wild type, respectively. In reactions with Mn, these three mutant enzymes also had comparable DNA synthetic processivity and K(m) values for primer terminus as the wild type enzyme (Table 1). These kinetic parameters thus render the mutant enzymes with specific activities comparable with the wild type enzyme in reactions with Mn.

Since mutation of Lys to Arg or Ala but not Asn increased the mutant's affinity (decreased the K(m)) for primer terminus in reactions with Mg as the metal activator, we also investigated the effect on template interaction when the positively charged side chain of this lysine residue is replaced by either a larger size charged side chain or is completely abolished. Inhibition by single-stranded DNA was compared, and no apparent difference was found between wild type and all three mutant enzymes (data not shown). This result indicates that this lysine residue is not involved in template interaction.

Results of these kinetic parameter studies suggest that mutation of this highly conserved Lys residue primarily affects the affinity for the incoming dNTP substrate and does not affect catalysis.

Effect of Metal Activator

The observed differences in these mutant enzymes' kinetic parameters in reactions with Mg from that of Mn led us to investigate these mutant enzymes' preference of metal activator. The optimal concentrations of each metal activator for the wild type and the three mutant enzymes were compared and are shown in Fig. 2. Like what we observed before (9) , the wild type enzyme prefers to utilize Mg as the metal activator with an optimal concentration at 10 mM and utilizes Mn as metal activator poorly with an optimal concentration at approximately 0.5 mM. In contrast, all three Lys mutant enzymes were able to utilize Mn as metal activator more effectively than the wild type enzyme (Fig. 2). Mutant enzymes K950R and K950N had similar optimal concentrations for Mg and Mn. Mutant enzyme K950A displayed a lower (42%) specific activity in reactions with Mn as compared with that with Mg (Fig. 2C), whereas mutant enzyme K950R had 70% of the specific activity in reaction with Mn as compared with reaction with Mg, and mutant enzyme K950N had a higher specific activity in reaction with Mn than with Mg ( Table 1and Fig. 2, B and D).


Figure 2: Metal titration assays for the wild type and mutant enzymes. Reactions were carried out as standard DNA synthesis reactions with varying concentrations of MgCl(2) (circle--circle) or MnCl(2) (--). Titration curves are plotted as specific activity of enzyme versus metal ion concentration for each enzyme.



Misinsertion Fidelity

Given the ability of these three Lys mutant enzymes to utilize Mn as the metal activator for catalysis like that of D1002N and T1003S in region I(8, 9) , we tested the misinsertion fidelity of these three Lys mutants in reactions with either Mg or Mn as the metal activator. Using a standing start primer-template (see ``Experimental Procedures''), we tested the incorporation of correct dTTP versus incorrect dCTP in reactions with Mg as the metal activator. We found that the misinsertion efficiency of the three Lys mutants was identical with that of the wild type. In contrast, in reactions with Mn, our results show that mutations of Lys had improved misinsertion fidelity over the wild type enzyme like that of the D1002N and T1003S mutant enzymes (8) (Table 2). Mutant enzymes K950R and K950A had 6- and 9.6-fold improved misinsertion fidelity compared with the wild type, while mutant enzyme K950N showed a >1900-fold improved misinsertion fidelity over the wild type. Thus, the three Lys mutant enzymes are metal-induced anti-mutators and the side chain of Lys, like that of Asp and Thr(8) , might have a role in the Mn-induced infidelity during DNA synthesis by DNA polymerase alpha.



LysSide Chain Has a Role in the Active Site and Is Involved in Interacting with dNTPs

Our finding of mutations of Lys to Arg, Ala, or Asn affecting apparent K(m) values for dNTP in reactions with either Mg or Mn as the metal activator suggests that the side chain of Lys may have a role in active site interacting with the incoming dNTP substrate. We, thus, used an inhibitor and several dNTP structural analogs to verify this notion.

We first tested the effect of an inhibitor, aphidicolin, on these three Lys mutant enzymes. Aphidicolin is a general inhibitor for all three major cellular alpha-like DNA polymerases, alpha, , and (3) . Aphidicolin acts as a competitive inhibitor of pyrimidine deoxynucleoside triphosphate, but, structurally, aphidicolin is not an analog of dNTPs. We have previously proposed a model of how aphidicolin forms hydrogen bonds with the purine base of the nucleotide in template in the active site of alpha-like DNA polymerases(5) . To test if Lys plays a role in the active site in interacting with metal activator(s) or the dNTP-metal activator complex, we tested the inhibitory effect of aphidicolin on the three mutant derivatives of Lys and compared their 50% inhibition point to that of the wild type reactions with Mg as metal activator. The three Lys mutant enzymes, K950R, K950A, and K950N, were 10, 14, and 33 times more sensitive to aphidicolin inhibition than the wild type enzyme, respectively (Table 3). These results suggest that Lys functions in the active site and aphidicolin affects the interaction between the Lys side chain and the dNTP substrate.



We next compared the three Lys mutant derivatives to wild type enzyme for their 50% inhibition points by an analog of dGTP, BuPdGTP, and its alpha,beta-methylene derivative, BuPdGMPCH(2)PP (Table 3). All three Lys mutant derivatives showed higher sensitivity to both of these compounds than the wild type enzyme. Mutant enzyme K950R had about 3 times higher sensitivity to both BuPdGTP and BuPdGMPCH(2)PP. Mutant enzyme K950A and mutant enzyme K950N both showed much higher sensitivity to the inhibition by BuPdGTP and BuPdGMPCH(2)PP than did the K950R (Table 3). These indicate that the Lys side chain indeed has a role in the active site and is involved in interacting with the incoming dNTP.

Results of the kinetic and inhibitor studies suggest that the positively charged side chain of Lys has a function in the active site and is involved in interacting with the dNTP-metal activator complex.

Structure Feature of dNTP Recognized by Lys

We next investigated what structural feature of dNTP interacts with the Lys side chain. The finding that all three Lys mutant derivatives have misinsertion fidelity efficiency identical with the wild type enzyme in reactions with Mg as the metal activator (Table 2) rules out the possible interaction of Lys with the nucleoside base moiety of the dNTP substrate. The abilities of all three Lys mutant enzymes to use Mn as metal activator effectively suggest that the structural feature of dNTPs interacting with the Lys side chain are either the deoxyribose sugar or the phosphate groups. We therefore systematically tested the interaction between these structural features of dNTP and the Lys side chain.

Does the LysSide Chain Recognize the Deoxyribose Sugar of the Incoming dNTP Substrate?

Two dCTP analogs with modifications in the deoxyribose sugar moiety, araCTP and ddCTP, were used to test their 50% inhibitory point (IC) on the three Lys mutant enzymes and compared to the wild type enzyme (Table 4). All three Lys mutant enzymes showed a higher resistance to araCTP inhibition than the wild type enzyme did. In contrast, all three Lys mutant enzymes showed higher sensitivity to ddCTP inhibition than the wild type enzyme. Mutant enzyme K950A with the entire positively charged side chain abolished had a 64-fold increase in its sensitivity to ddCTP and was 7-fold more resistant to araCTP than the wild type enzyme (Table 4). Thus, alteration of the furanose ring conformation of a dNTP has an effect on the interaction between a dNTP and the Lys side chain. Since alterations of the deoxyribose sugar also affects the orientation of the triphosphate group of dNTP, we, therefore, analyzed whether the alpha-, beta-, or -phosphate group was the structural feature of a dNTP recognized by the Lys side chain.



Does the LysSide Chain Recognize the beta- or -Phosphate Group of the Incoming dNTP Substrate?

We tested whether the Lys side chain has a role in properly positioning the triphosphate moiety of the incoming dNTP for metal activator chelation. Interaction between the phosphate groups of the dNTP substrate and the positively charged side chain of Lys could also facilitate a nucleophilic attack of the incoming primer 3`-hydroxyl group for deoxynucleotidyl transfer. We, thus, investigated the effect of an altered phosphate group of dNTP on the reactivities of the three Lys mutant enzymes and compared them to the wild type. In our inhibition studies of BuPdGTP and BuPdGMPCH(2)PP (Table 3), the patterns and the extent of inhibition for the three Lys mutant enzymes compared with the wild type enzyme were similar. Since the structural difference between these two compounds is the substitution of the oxygen group on beta-phosphate of BuPdGTP with methylene (-CH(2)-), the result suggested that modification of the beta-phosphate group does not have any effect on the interaction between the Lys side chain and the incoming dNTP substrate. To verify this observation, we tested the effect of pyrophosphate (PP(i)) and two analogs of PP(i), carbonyldiphosphonate and phosphonoacetic acid, for their 50% inhibition point (IC). The three mutant enzymes were inhibited by pyrophosphate to an extent similar to the wild type enzyme (Table 4). Inhibition patterns of the three mutant enzymes and the wild type enzyme by the two pyrophosphate analogs were variable (Table 4). However, like the inhibition studies with araCTP and ddCTP, K950A, with the Lys side chain abolished, showed the highest sensitivity to carbonyldiphosphonate and phosphonoacetic acid, as well as to BuPdGTP and BuPdGMPCH(2)PP. This suggests that the side chain of Lys might be involved in interacting with the triphosphate moiety of the incoming dNTP substrate. Since the three mutant enzymes showed similar if not identical sensitivity to pyrophosphate, BuPdGTP, and its beta-phosphate analog BuPdGMPCH(2)PP as the wild type enzyme, the beta- and -phosphates of the incoming dNTP are not likely to be the structural feature directly interacting with the Lys side chain.

Does the LysSide Chain Interact with the alpha-Phosphate Group of the dNTP?

The finding that these three Lys mutant enzymes could utilize Mn more effectively than the wild type enzyme, had altered sensitivity to araCTP and ddCTP, and were insensitive to pyrophosphate inhibition led us to investigate whether the alpha-phosphate group of the incoming dNTP is the structural feature recognized by the Lys side chain. We compared the affinity difference of the three Lys mutant enzymes for an alpha-phosphate analog, dCTPalphaS, and for dNTP (Table 5) in reactions with either Mg or Mn as the metal activator. The dCTPalphaS used in this study is the S(p) diastereomer which has been documented to be an active substrate for E. coli polymerase I(19) .



In reactions with Mg, wild type enzyme had 22-fold higher affinity for dTTP than dCTPalphaS (Table 5). Mutant enzyme K950R had 3-fold higher affinity for dCTPalphaS than dNTP (K(m)^S = 86 ± 46 µM and K(m)^O = 250 ± 43 µM), but comparable affinity for dCTPalphaS as the wild type enzyme (K(m)^S = 86 µM ± 46 µM for K950R and K(m)^S = 74 ± 12 µM for the wild type enzyme). Mutant enzyme K950A had 5- to 6-fold lower affinity to dCTPalphaS than the normal dNTP; mutant enzyme K950N had 13-fold lower affinity for dCTPalphaS than dNTP (Table 5). Mutant enzymes K950A and K950N had 6 and 24 times lower affinity (higher K(m) values) for dCTPalphaS than the wild type enzyme. These results indicate that substitution of the oxygen moiety of the alpha-phosphate by sulfur profoundly affects the affinity of the Lys mutant enzymes for the alpha-phosphate analog. This suggests that the Lys side chain interacts with the alpha-phosphate group of the incoming dNTP substrate.

The LysSide Chain Interacts with the Oxygen Moiety of the alpha-Phosphate of dNTP Substrate

We then compared the three mutants with the wild type enzyme for their affinity ratio for dCTPalphaS to dNTP (K(m)^S/K(m)^O). Mutant enzyme K950N with a polar amide side chain replacing the -amino side chain had less than a 2-fold lower affinity ratio for dCTPalphaS to dTTP as compared with that of the wild type (K(m)^S/K(m)^O = 13 for K950N, and K(m)^S/K(m)^O = 22 for the wild type enzyme) (Table 5). In contrast, by replacing the -amino side chain of Lys with a larger positively charged side chain, the mutant enzyme K950R showed a 73-fold lower affinity ratio of dCTPalphaS to dTTP than the wild type enzyme (K(m)^S/K(m)^O = 0.3 for K950R compared to the wild type enzyme of K(m)^S/K(m)^O = 22). Mutant enzyme K950A with the -amino side chain abolished had an approximately 4.5-fold lower affinity ratio for dCTPalphaS to dTTP than the wild type enzyme (K(m)^S/K(m)^O = 5.0 for K950A compared to K(m)^S/K(m)^O = 22 for the wild type enzyme).

In contrast to the reactions with Mg as the metal activator, in reactions with Mn as metal activator, all three mutant enzymes as well as the wild type enzyme had lower affinity (higher K(m) values) for dCTPalphaS than the wild type enzyme. Furthermore, the three mutant enzymes and the wild type enzyme had comparable or equal affinity ratio of dCTPalphaS to dTTP (K(m)^S/K(m)^O) (Table 5).

We then compared the three mutant enzymes to the wild type enzyme for their catalysis rate (k) in utilizing dCTPalphaS versus dNTP as substrate. In reactions with Mg, the wild type enzyme had 2.6-fold higher k in utilizing normal dNTP versus dCTPalphaS as substrate with a ratio of k^S/k^O of 0.38. Mutant enzyme K950R did not show a significant difference in its k value when either normal dNTP or dCTPalphaS was used as substrate. Mutant enzyme K950A like the wild type enzyme had an approximately 2-fold higher k when dNTP was used as substrate than when dCTPalphaS was used as substrate (k^S = 0.68 and k^O = 0.27). Mutant enzyme K950N, interestingly, had higher k when dCTPalphaS was used as substrate versus dNTP (Table 5).

When Mn was used as the metal activator, the wild type enzyme had identical k values in utilizing either dCTP or dCTPalphaS as a substrate. Mutant enzymes K950R and K950N both had comparable k values in using dCTPalphaS or normal dNTP as a substrate. Mutant enzyme K950A showed a 2-fold lower k in using dCTPalphaS as the substrate than with dCTP as the substrate, like that observed in the Mg-catalyzed reaction.

By comparing the affinity (K(m)) and catalysis (k) of these mutant enzymes to the wild type enzyme for utilizing dNTP and dCTPalphaS as substrate, it is apparent that substitution of the -P=O by -P=S in the alpha-phosphate group of dCTP profoundly affects the affinity of the enzyme's binding to the incoming dNTP substrate, but does not significantly affect the rate of catalysis.

These results together with the findings that the three Lys mutant enzymes are able to utilize Mn as metal activator strongly suggest that the positively charged side chain of Lys either directly or indirectly participates in interactions with the oxygen moiety of the alpha-phosphate group of the incoming dNTPs, either to position the dNTP to interact with the metal activator or to facilitate the nucleophilic attack by the 3`OH group of the incoming primer.


DISCUSSION

We have used the recombinant human DNA polymerase alpha as the prototypic model for the three principal cellular DNA polymerases alpha, , and to elucidate the functional roles of several highly invariant amino acid residues in the active site. We altered several invariant residues by site-directed mutagenesis based on the rationale that we described(20) . We generated a panel of mutants that did not have any detectable gross alteration of the protein structure(4, 5, 9, 20) . Thus, it is reasonable to assume that the structural alterations resulting from each mutation are confined to the position of the mutated side chains. By steady-state kinetic analysis of the mutant enzymes, we have defined the functions of several residues in the two most conserved regions (regions I and II) of the active site(4, 5, 8, 9) . In this report, we have extended our studies of the active site by investigating the function of a highly invariant lysine residue in the third most conserved region (Fig. 1).

Previous study has documented that the interaction of DNA polymerase alpha with its substrates obeys a rigidly ordered sequential terreactant mechanism, with template as the first substrate, followed by primer as the second substrate and dNTP as the third. Specification of which of the four dNTPs has kinetically significant binding is determined by the base sequence of the template(21) . A similar ordered sequential terreactant mechanism was also proposed by Dahlberg and Benkovic (22) for the Klenow fragment of E. coli polymerase I. Given the universal ordered sequential mechanism for both eukaryotic DNA polymerase alpha and prokaryotic E. coli polymerase I, and depending on the base sequence of the template, DNA polymerases have different modes of interaction with the incoming dNTP. Studies of Klenow fragment have shown a rate difference in using dTTP versus dGTP(23) . In this study, we did not compare the difference of either the wild type or the mutant enzymes for their affinity (K(m)) in binding each different incoming dNTP or the difference in binding purine versus pyrimidine deoxyribose triphosphate. It is possible that the side chain of this highly invariant Lys of alpha-like DNA polymerases like that of E. coli polymerase I has a different mode of interaction with each different incoming dNTP(23) . Here, we assume in the enzyme where the protein contacts the dNTP, the Lys side chain has the same interaction with all four of the dNTPs in all circumstance. We also only evaluated the side chain function of Lys by kinetic analysis in the state of catalytically competent ternary complex at the point of phosphodiester bond formation and pyrophosphate release.

Mutations of LysSide Chain Affect dNTP Affinity and Metal Activator Utilization

In this study we have observed the following. (i) Mutation of Lys to Arg, Ala, and Asn has an effect on the mutant enzyme's K(m) for dNTP in reactions with either Mg or Mn as metal activator, but has only a moderate effect on their k. This implies that the side chain of Lys is mainly involved in interacting with the dNTP substrate and not in catalysis. In addition, inhibition studies with an active site inhibitor and analogs have further verified that the side chain of Lys has a role in the active site interacting with a dNTP substrate. (ii) Mutations of this highly conserved lysine residue allows the mutant enzymes to utilize Mn as metal activator more efficiently than the wild type enzyme (Fig. 2) as observed in mutations of Asp and Thr in region I of human DNA polymerase alpha(9) . However, a noteworthy difference between the Lys mutant enzymes and the Asp and Thr mutant enzymes is that mutations of Asp and Thr have a profound effect on the enzyme's catalysis k, whereas mutations of Lys mainly affect the mutant enzymes' K(m) values and have only a mild effect on their catalysis (k) (Table 1).

Metal Activator Effect

Despite the fact that mutations of Lys affect the K(m), none of the Lys mutant derivatives displayed significant differences in their misinsertion efficiency as compared to wild type enzyme when Mg was used as the metal activator (Table 2). This suggests that in a polymerase alpha-Mg complex, the side chain of Lys does not interact with the nucleotide base directly and is not responsible for pairing the incoming dNTP to the appropriate nucleotide base in the template. In reactions with Mn, all three Lys mutants showed an improved misinsertion fidelity (Table 2) like that observed in mutants D1002N and T1003S of human polymerase alpha region I(8) .

Studies of E. coli polymerase I have proposed that there is an indirect interaction between metal activator and the deoxyribose of dNTP(24) . It has been proposed that E. coli polymerase I-Mg complex selectively prefers the C2`-endo conformation of the deoxyriboside of dNTPs, while the polymerase I-Mn complex is less selective for this conformation. Thus, in the Mn-catalyzed reaction, the deoxyribose freely equilibrates between the C2`- and C3`-endo conformations of the deoxyriboside. It is possible that the polymerase alpha-Mn complex like that of E. coli polymerase I-Mn complex, is less selective for C2`-endo conformation of deoxyriboside and allows the sugar ring to freely equilibrate between C2`- and C3`-endo conformations. This might enhance the stringency for the polymerase alpha-Mn complex in its specification for a correct dNTP over an incorrect dNTP resulting in improved misinsertion fidelity.

Based on structural data together with mutagenesis studies of Klenow fragment, Joyce and Steitz and co-workers (12, 25) have proposed a possible mechanism for the polymerase reaction in which two metal ions are involved in mediating catalysis. In the active site of a DNA polymerase, several carboxylate side chains, such as the Asp and Asp of the Klenow and Asp and Asp of human polymerase alpha, function to anchor two divalent metal ions (Mg) for catalysis. One Mg promotes the deprotonation of the 3`-hydroxyl of the primer, while the second Mg facilitates the formation of the pentacovalent transition state at the alpha-phosphate of the dNTP and the loss of pyrophosphate. Since mutations of Lys affect both the metal activator utilization and affinity for dNTP, the side chain of Lys therefore might interact either directly or indirectly with the second metal ion chelated alpha-phosphate of the incoming dNTP.

LysSide Chain Interacts with Oxygen Moiety of the alpha-Phosphate of the dNTP Substrate

The abilities of the three Lys mutant enzymes to utilize Mn as metal activator more efficiently than the wild type enzyme suggest that the positively charged lysine side chain may have an influence on the configuration of the negatively charged phosphate groups of the incoming dNTP. Comparison of the inhibitory effect of pyrophosphate and the inhibitory effect of BuPdGTP versus BuPdGMPCH(2)PP on the wild type enzyme and the three mutant enzymes have shown that the beta- and -phosphates are not likely to be in direct contact with the lysine side chain ( Table 3and Table 4). Analogs of dNTP containing alterations in the ribose moiety had notable effects on the reactivity of the three Lys mutant enzymes as compared to the wild type enzyme (Table 4). Mutant enzyme K950A which has the positive charge side chain abolished always displays higher sensitivity or resistance to the dNTP analogs regardless of whether the analog has deletion of the 3`-OH group as in ddCTP or has a twisted deoxyribose ring as in araCTP(26) .

Since alteration of the deoxyribose in either araCTP or ddCTP could also affect the orientation of the oxygen group of alpha-phosphate, we also tested the interactions between the Lys side chain and the oxygen group of alpha-phosphate with an analog, dCTPalphaS, in reactions utilizing either Mg or Mn as metal activator. We compared each enzyme's affinities (K(m)) and catalysis (k) for utilizing dCTPalphaS versus dNTP as substrate in reactions with either Mg or Mn (Table 5). We also compared each enzyme's affinity ratio for dCTPalphaS versus dNTP (K(m)^S/K(m)^O) with the wild type enzyme. In reactions with Mg, mutation of the Lys side chain by either replacing it with a larger charged side chain (K950R) or abolishing the charged side chain (K950A) had a significant effect on the mutant enzyme's affinity to dCTPalphaS versus dNTP substrate. In contrast, replacing the -amino side chain of Lys to Asn (K950N) appears not to affect the mutant's affinity ratio for dCTPalphaS versus dNTP. In Mn-catalyzed reactions, all three mutants showed comparable affinity ratio, K(m)^S/K(m)^O, as the wild type enzyme. We reason that this difference in metal effect might be due to the polymerase alpha-Mn complex having less selective preference for C2`-endo deoxyribose. The presence of the C3`-endo form of dNTP could affect the affinity between the dNTP and the side chain of Lys resulting in an observed mild effect on the affinity of the mutant enzymes for dCTPalphaS.

In sum, results presented in this study strongly suggest that the structural feature of the incoming dNTP recognized by the positively charged Lys side chain is the oxygen moiety of the alpha-phosphate group.

A Proposed Model of How Active Site Residues Collaborate for Polymerase Catalysis

There is no structural data for any member of the alpha-like DNA polymerases (family B). However, in light of the primary sequence conservation of these three highly invariant regions among all of the alpha-like DNA polymerases and the similarities between the predicted secondary structure of these three regions in alpha-like DNA polymerases with the crystal structures of Klenow fragment and HIV-1 reverse transcriptase(10, 11) , it is reasonable to assume that these three highly conserved regions in the alpha-like polymerases are components of the active site. The Lys described in this report is located in a predicted alpha-helix. This helix might be positioned in the active site like the O-helix of Klenow fragment(10) . In the active site of Klenow fragment, binding of the incoming dNTP is not identical under all circumstances for each dNTP(23) . Studies of site-directed mutations of a residue Arg in the O-helix of Klenow fragment suggested that Arg contacts the beta- or -phosphate of the incoming dNTP substrate. When the incoming dNTP is dGTP, in addition to Arg, two residues in the O-helix, Lys and Phe, also participate in the binding(23) . Without a physical structure of the alpha-like polymerases, we cannot assume that Lys of polymerase alpha is equivalent to the Lys of the Klenow fragment. However, it is interesting that both family A and family B polymerases have a highly conserved lysine residue located in an alpha-helix, and both appear to be involved in interacting with the incoming dNTP substrate.

Our mutational studies (4, 5, 8, 9) have identified the functions of side chains of several amino acid residues localized in the active site of the alpha-like polymerases. The results have supported a model of the active site of alpha-like polymerases. In human polymerase alpha, Asp and Asp, and Thr located in an anti-parallel beta-sheet (region I) chelate with the metal activator cation, Mg, which in turn chelates to the oxygen moiety of beta- and -phosphate of the incoming dNTP(8, 9) . The phenyl ring side chain of Tyr (in region II) interacts with the nucleotide base of the incoming dNTP to properly position the incoming dNTP for Watson-Crick base pairing(5) . The oxygen moiety of the Ser hydroxyl side chain in region II forms a hydrogen bond either directly or indirectly with the 3`-OH terminus of the primer. The hydrogen bond formation might enhance the oxygen moiety at the 3`-OH-primer terminus for nucleophilic attack at the alpha-phosphate of the incoming dNTP(4) . The positively charged side chain of Lys located in an alpha-helix in region III of human polymerase alpha interacts either directly or indirectly with the oxygen group of the alpha-phosphate of the incoming dNTP. This interaction of a positively charged side chain will neutralize the negative charge on the alpha-phosphate to facilitate nucleophilic attack of the incoming primer 3`-hydroxy group. This proposed model of the alpha-like polymerase active site (Fig. 3) is based entirely on biochemical data and can only be verified in the future by crystallographic data of a ternary complex of polymerase alpha with primer-template and dNTP substrates.


Figure 3: Model of the alpha-like DNA polymerase active site residues collaborate for nucleotidyl transfer. Shown are the proposed functions of residues of human DNA polymerase alpha studied by site-directed mutagenesis. The three region I residues, Asp, Thr, and Asp, shown here bound to the metal-nucleotide complex are adopted from (9) . The phenyl ring of Tyr in region II that interacts with the nucleotide base moiety of the incoming dNTP and the hydroxyl side chain of the Ser residue that hydrogen bonds to the 3`-OH terminus of the primer shown here are adopted from (4) . The positive charged side chain of Lys of region III is shown here to interact with the oxygen group of the alpha-phosphate of the incoming dNTP. An X is shown here to depict the unknown side chain(s) of residue(s) in the active site that might participate in chelating the Mg metal activator.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA14835. 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. Tel.: 415-725-4907; Fax: 415-725-6902.

(^1)
The abbreviations used are: dCTPalphaS, deoxycytidine 5`-O-(1-thiotriphosphate); BuPdGMPCH(2)PP, N^2-(p-n-butylphenyl)-2`-deoxyguanosine 5`-(p^1, p^2-methylene)-triphosphate.


ACKNOWLEDGEMENTS

We thank Krista L. Conger for assistance in constructing the mutants and Dr. William C. Copeland and Dr. I. Robert Lehman for reading this manuscript and for helpful discussions.


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