629 DNA Polymerase Active Site MUTANTS IN CONSERVED RESIDUES Tyr254 AND Tyr3” ARE AFFECTED IN dNTP BINDING*

429 DNA polymerase shares with other a-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs ”D-NSLYP” and “K-NS(L/ V)YG,” regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100,27-38) have been proposed to be part of the polymerization active site of a-like DNA polymerases. One 429 DNA polymerase mutant in residue Tyr264, located in conserved region 1, and two mutants in residue Tyr3’0, located in conserved region 2a, have been characterized. The three 429 DNA polymerase mutant proteins were affected in polymerization when Mg2+-dNTPs were used as sub- strate. However, when the substrate was Mn2+-dNTP, mutants behaved as the wild-type 429 DNA polymer- ase. Mutant Tyr264 to Phe (Y254F) was strongly affected in the protein-primed initiation step of 429 DNA replication showing a decreased affinity for Me2+-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP drawn immediately after the DNA polymerization reaction. The chromatogram was developed with 0.5 M lithium formate (pH 3.0), conditions in which it is possible to separate 5'dAMP from the DNA and unincorporated dNTP. 5""'P-Labeled oligonucleotide SP1 and hybrid molecules between oligonucleotide SPlc+6 and 5'-"P-labeled oligonucleotide SP1 were also used as substrates for 3' + 5' exonuclease activity on ss- and dsDNA, respectively. The assay conditions were essentially as described for the polymerase/exonuclease coupled assay, but in the absence of dNTPs. After incubation for 2 min at 30 "C, the samples were analyzed by 8 M urea-20% polyacrylamide gel electrophoresis. Degradation of the 5'-''P-Iabeled primer (SP1) was detected by autoradiography. Densitometric scans of the exposed films were done for quantitation.

The linear dsDNA' of Bacillus subtilis phage 429 replicates by a protein-priming mechanism (Salas, 1991) in which a virally encoded DNA polymerase catalyzes both the formation of the TP-dAMP covalent complex (initiation reaction) and its further processive elongation to produce unit-length 429 * This investigation was supported in part by Research Grant 5R01 GM27242-12 from the National Institutes of Health, Grant PB90-0091 from Direcci6n General de Investigacibn Cientifica y Tecnica, Grant BIOT CT 91-0268 from European Economic Community, and an institutional grant from Fundaci6n Rambn Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
429 DNA polymerase was included in the group of a-like DNA polymerases because of its sensitivity to aphidicolin and to the nucleotide analogs BuAdATP and BuPdGTP (Blanco and Salas, 1986;Bernad et al., 1987), specific inhibitors of eukaryotic DNA polymerase a (Khan et al., 1984Butler et al., 1990). Another criteria to consider 429 DNA polymerase as an a-like DNA polymerase was the fact that it contains several regions of amino acid homology highly conserved in the C-terminal portion of a-like DNA polymerases (Larder et al., 1987;Bernad et al., 1987;Wong et al., 1988;Blanco et al., 1991;Ito and Braithwaite, 1991). It has been proposed that these conserved regions may play an important role in the function of the enzyme, being part of the active center for synthetic activities (Larder et al., 1987;Gibbs et al., 1988). This hypothesis has been supported by site-directed mutagenesis in 429 DNA polymerase; thus, point mutants in the most conserved regions, characterized by the amino acid motifs "D-NSLYP" (region l),"K-NS(L/V)YG" (region 2a), and "YGDTDS" (region 3) had normal 3' + 5' exonuclease activity but they were affected in the synthetic activities (proteinprimed initiation and/or DNA polymerization) (Bernad et al., 1990a(Bernad et al., , 1990bBlanco et al., 1991). In this paper we report a detailed characterization of purified 429 DNA polymerase mutants Y254F2 (region 1) and Y390S and Y390F (region 2a). The results obtained indicate that residues Tyr254 and Tyr3" are involved, directly or indirectly, in Me2+-dNTP binding.

Proteins
Restriction endonucleases, T4 DNA ligase, and polynucleotide kinase were from New England Biolabs. The site-directed mutagenesis kit, Muta-GeneTM, was from Bio-Rad. Sequenase version 2.0, from U. S. Biochemical Corp., was used for sequencing. Wild-type or $29 DNA polymerase mutants were purified essentially as described (Blanco and Salas, 1984) with the modifications to be published elsewhere. TP was purified as described . ' Mutations are indicated by original amino acid (in single-letter notation), its position, and the replacing amino acid, i.e. Y254F = Tyr254 + Phe.

Plasmids and Bacteria
Plasmid pT7-3W21, harboring the 629 DNA polymerase gene, was obtained as described (Bernad et al., 1989). The E.coli strain K514 was used as a host for transformation with pT7-3 (Tabor and Richardson, 1985) recombinants containing the $29 DNA polymerase gene. The E.coli X lysogen BL21(DE3) strain (Studier and Moffatt, 1986) was used for expression of the mutant proteins.

Site-directed Mutagenesis and Expression of $29 DNA Polymerase Mutants
Residues T y P 4 and Tyr390 of $29 DNA polymerase were selected as targets for site-directed mutagenesis because these residues are among the most conserved ones in each of the two motifs D-NSLYP and K-NS(L/V)YG of a-like DNA polymerases (Blanco et al., 1991); in addition, these two motifs show significant amino acid crosssimilarity. The changes were designed taking into account secondary structure predictions (Chou andFasman, 1978 Garnier et al., 1978) and the proposals for conservative changes made by Argos (1987). The wild-type $29 DNA polymerase gene cloned into M13 mp19 (M13 mp19W21) (Bernad et al., 1989), was used for site-directed mutagenesis, carried out essentially as described (Kunkel, 1985b;Kunkel et al., 1987). The presence of the desired mutations and the absence of any other changes were confirmed by complete sequencing of each 429 DNA polymerase mutant gene. For expression, the fragments carrying the different mutations were cloned in plasmid pT7-3W21, which expresses $29 DNA polymerase under the control of the T7 RNA polymerase specific 610 promoter (Tabor and Richardson, 1985). Expression of the mutant proteins was carried out in the Escherichia coli strain BL21(DE3), which contains the T7 RNA polymerase gene under the control of the lacUV5 promoter, and thus ranoside (Studier and Moffatt, 1986). it is inducible by the lactose analog isopropyl-1-thio-P-D-galactopy-

DNA Manipulations
Plasmid DNA minipreparations (Birnboim and Doly, 1979) were direct substrates for sequencing by the dideoxy chain-termination method (Sanger et al., 1977). A set of synthetic oligonucleotides complementary to $29 DNA polymerase gene sequences were used as sequencing primers.

Initiation Assay (TP-dAMP Complex Formation)
The reaction mixture contained, in 25 pI, 50 mM Tris-HC1 (pH 7.5), 1 mM MnC12, 20 mM ammonium sulfate, 0.1 mg/ml BSA, 1 mM dithiothreitol, 4% glycerol, 50 mM NaCl, 0.25 p M [a-32P]dATP, the indicated amounts of purified TP and either wild-type or mutant 429 DNA polymerase and, when indicated, TP. DNA (0.5 pg) as template. The nucleotide analogs BuAdATP or BuPdGTP were used when indicated. After incubation for the indicated time and temperature the reaction was stopped by adding up to 10 mM EDTA, 0.1% SDS, the samples were filtered through Sephadex G-50 spin columns in the presence of 0.1% SDS, and the excluded volume was analyzed by SDS-polyacrylamide gel electrophoresis as described (Peiialva and Salas, 1982). Quantitation was done either by excising from the dried gel the radioactive band corresponding to the TP'dAMP complex and counting the Cerenkov radiation, or by densitometric scans of the exposed films.

DNA Polymerase Assay (Filling-in Reaction)
The incubation mixture contained, in 25 pl, 50 mM Tris-HC1 (pH 7.5), either 10 mM MgC12 or 1 mM MnC12, 1 mM dithiothreitol, 4% glycerol, 50 mM NaCI, 2.5 ng of either wild-type or mutant $29 DNA polymerase, 0.5 pg of EcoRI-digested $29 DNA or BamHI-digested pUC19 DNA as templates, and either 0.25 p~ [cY-~'P]~ATP and 200 pM each dGTP and dTTP, or 0.25 p M [a-32P]dGTP and 200 p~ each dATP and dTTP, respectively. When indicated, BuAdATP or Bu-PdGTP were added. After incubation for 2 min at 30 "C, the reaction was stopped by adding up to 10 mM EDTA, 0.1% SDS and the samples were filtered through Sephadex G-50 spin columns as described above. The excluded volume was counted (Cerenkov radiation) and analyzed by agarose gel electrophoresis and autoradiography. Densitometric scans of the exposed films were done for quantitation.

Processive DNA Polymerase Assays
Replication of Primed-MI3 DNA-The incubation mixture contained, in 25 pl, 50 mM Tris-HC1 (pH 7.5), either 10 mM M&l2 or 1 mM MnC12, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml BSA, the indicated concentrations of each dNTP and 0.25 p M [a-32P]dATP, 0.25 pg of primed-M13 mp2 ssDNA and 20 ng of either wild-type or mutant $29 DNA polymerase. When indicated, BuAdATP or Bu-PdGTP were added. After incubation for 30 min at 30 "C the reactions were stopped by adding up to 10 mM EDTA, 0.1% SDS, the samples were filtered through Sephadex G-50 spin columns in the presence of 0.1% SDS, and the excluded volume was analyzed by alkaline 0.7% agarose gel electrophoresis and autoradiography. Densitometric scans of the exposed films were done for quantitation.
Replication of TP-DNA-The incubation mixture contained, in 25 pl, 50 mM Tris-HCI (pH 7.5), either 10 mM MgCl, or 1 mM MnCI2, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml BSA, 20 mM ammonium sulfate, the indicated concentration of each dNTP and 0.25 p~ [a-32P]dATP, 0.5 pg of TP.DNA , 20 ng of either wild-type or mutant $29 DNA polymerase, and 20 ng of TP. When indicated, BuAdATP or BuPdGTP were added. After incubation for 5 min at 30 "C the reactions were stopped by adding up to 10 mM EDTA, 0.1% SDS, filtered as above and the Cerenkov radiation counted. Samples were subjected to alkaline 0.7% agarose gel electrophoresis and autoradiography.

Polymerase/Exonuclease Coupled Assay
Hybrid molecules between oligonucleotide SPlc+6 and 5'-32Plabeled oligonucleotide SP1 (described before) were obtained. This hybrid, containing 5"protruding ends, can be used both as substrate for the 3' --f 5' exonuclease activity and as template-primer for DNA polymerization. The incubation mixture contained, in 25 pl, 50 mM Tris-HC1 (pH 7.5), 10 mM MgC12, 1 mM dithiothreitol and 10 ng of either wild-type or mutant $29 DNA polymerase. After incubation for 5 min at 30 "C the reactions were stopped by addition of EDTA to 10 mM. Samples were analyzed by 8 M urea, 20% polyacrylamide gel electrophoresis and autoradiography. Polymerization or exonuclease activities is detected as an increase or decrease, respectively, in the size of the 5"labeled primer-strand (SP1) (Garmendia et al., 1992).

3' --f 5' Exonuclease Assays
When 3"labeled EcoRI-digested 429 DNA, obtained as indicated before, was used as substrate, the incubation mixture contained, in 10 pl, 50 mM Tris-HC1 (pH 7.5), 10 mM MgC12, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml BSA, 2.5 ng of either wild-type or mutant 429 DNA polymerase and when indicated, BuAdATP or BuPdGTP. After incubation for 2 min at 25 "C the reactions were stopped by adding up to 10 mM EDTA. Released 3ZP-dAMP was determined by polyethylenimine-cellulose thin layer chromatography and autoradiography. The chromatogram was developed with 0.5 M PO~HZK, conditions in which dAMP (fast migrating spot) and DNA (remaining at the origin) can be separated. After autoradiography, quantitation was done by densitometry of the [32P]dAMP spot relative to the total amount of 32P-labeled substrate.
When indicated, the dAMP turnover coupled to the filling-in assay described before was determined by polyethylenimine-cellulose thin layer chromatography and further autoradiography of samples with-drawn immediately after the DNA polymerization reaction. The chromatogram was developed with 0.5 M lithium formate (pH 3.0), conditions in which it is possible to separate 5'dAMP from the DNA and unincorporated dNTP.
5""'P-Labeled oligonucleotide SP1 and hybrid molecules between oligonucleotide SPlc+6 and 5'-"P-labeled oligonucleotide SP1 were also used as substrates for 3' + 5' exonuclease activity on ss-and dsDNA, respectively. The assay conditions were essentially as described for the polymerase/exonuclease coupled assay, but in the absence of dNTPs. After incubation for 2 min a t 30 "C, the samples were analyzed by 8 M urea-20% polyacrylamide gel electrophoresis. Degradation of the 5'-''P-Iabeled primer (SP1) was detected by autoradiography. Densitometric scans of the exposed films were done for quantitation.

Mutations Y254F, Y390S, and Y390F Affect the Affinity for dNTPs
Mutant Y254F in region I is affected in TP. dAMP initiation complex formation.-The ability of the different mutants to form the initiation complex TP.dAMP was assayed using TP.DNA as template. Mutant Y254F had been shown to be greatly affected in TP.dAMP initiation complex formation in the presence of Mg2+, the activity being 3% of that of wildtype 429 DNA polymerase, whereas mutants in region 2a, Y390F and Y390S, were only slightly affected (Blanco et al., 1991). Using Mn2+ as metal activator , mutant Y254F had about 12% of the wild-type activity ( Fig.  1A) and mutants in Tyrsg" were only slightly affected (not shown). The apparent affinity for TP was found to be the same for both wild-type and mutant Y254F 429 DNA polymerases when assayed in the template-independent deoxynucleotidylation of TP (see below) using different amounts of the latter (results not shown). The V,,, values in the formation of the TP.dAMP complex of wild-type and mutant 429 DNA polymerases were found to be approximately the same (not shown), ruling out a defect of the Y254F mutant in the catalysis of the initiation reaction. However, the apparent K, value for M$+-dATP in this reaction was found to be higher in mutant Y254F (150 y~) than in wild-type 429 DNA polymerase (10 p~) . When the substrate was Mn2+-dATP, the K , value for DNA polymerase mutant Y254F was 4 y~ and that of the wild-type was 1 p~. All these data indicate that mutation in Tyr254 affects the affinity for Me2+-dATP in the initiation reaction.
We have recently shown that 429 DNA polymerase is able to catalyze the covalent linkage of any of the four dNTPs to TP in the absence of template. In the presence of Mn2+ and a t low dATP concentration (0.25 y~) , the efficiency of this reaction is about 0.3% that of the reaction in the presence of the natural template, TP.DNA . Fig. 1B shows that the formation of TP. dAMP complex catalyzed by DNA polymerase mutant Y254F in the absence of template was also strongly reduced, the activity being 14% that of wildtype 429 DNA polymerase. Since this reaction does not re- B, no template was added; 120 ng of TP and 100 ng of the indicated $29 DNA polymerase were used. Incubation was for 2 h a t 25 "C. quire template, this result indicates that a defective template binding is not responsible for the mutant phenotype. The relative usage of the four dNTPs by either wild-type or mutant Y254F 429 DNA polymerases can be studied in this templateindependent reaction. For the wild-type enzyme, the relative dNTP usage, probably as a consequence of the affinity of each nucleotide for the dNTP binding site, was d T T P > dATP > dGTP> dCTP ; see also Fig. 2 A ) . However, in the case of mutant Y254F, dATP was the preferred substrate, the pattern of relative usage being dATP > d T T P > dGTP > dCTP (Fig. 2B) 3A). However, all the mutant proteins showed a turnover (exonucleolysis/polymerization) higher than that of wild-type 429 DNA polymerase (Fig. 3B), in agreement with previous data (Blanco et al., 1991). On the contrary, when Mn2+ was used in the filling-in reaction all mutant 429 DNA polymerases showed an activity similar or even higher (mutant Y254F) than that of the wild-type enzyme (Fig. 3C) and the coupled turnover was also similar in wild-type and mutant proteins (Fig. 3 0 ) . Mutants Y254F, Y390S, and Y390F Have a Decreased Affinity for Mg2+-dNTPs-Two kinds of processive replication assays were carried out: TP.DNA replication and primed-M13 DNA replication. Previous results indicated that mutants Y254F and Y390F were greatly affected in TP .DNA replication when the assay was carried out in the presence of Mg2+ ions at 40 p~ dNTP, whereas mutant Y390S behaved almost like wild-type 429 DNA polymerase under these conditions (Blanco et al., 1991). Fig. 4A shows that, in the presence of Mg2+-dNTPs, the wild-type/mutant ratio of TP.
DNA replication activity was dependent on the dNTP concentration used. Even mutant Y390S, which at high dNTP concentration (100 p~) behaved as wild-type 429 DNA polymerase, had very low activity at 10 p~ dNTP. In agreement with the results obtained for the filling-in reaction, when Mn2+-dNTPs were used instead of M$+-dNTPs, the effect of DNA was used as template (Fig. 4B), in the presence of M e -dNTPs, replication was greatly affected with DNA polymerase mutants Y390S and Y254F using dNTP concentration up to 20 p~, and mutant Y390F showed no activity. Again, when Mn"-dNTPs were used, the effect of the mutations was overcome (Fig. 4B). These data suggest that the affinity for Mg2+-dNTP in all mutant 429 DNA polymerases is lower than that of the wild-type enzyme. d N T P Concentration-dependent Balance between Synthesis and Degradation by Wild-type and Mutant 429 DNA Polymerases-A dynamic equilibrium between 3' + 5' exonuclease activity and polymerase activity, probably due to the competition between the corresponding active sites for binding the 3'-end of the template, has been recently demonstrated for 429 DNA polymerase; thus, for the wild-type enzyme, a minimal dNTP concentration is necessary for polymerization to compete out exonucleolysis (Garmendia et al., 1992). We have used SPl/SPlc+G hybrid as template with either wild-type or mutant 429 DNA polymer'ases in the presence of M$' and increasing amounts of dNTPs, to determine the dNTP concentration needed to produce the first and subsequent polymerization events on that template. Fig. 5A shows that for wildtype 429 DNA polymerase, the "lP-labeled primer strand (SP1,15-mer) of the hybrid oligonucleotide SPl/SPlc+G (see "Materials and Methods") underwent degradation or polymerization, depending on the dNTP concentration. Mutant $29 DNA polymerases Y254F ( B ) , Y390S (C), and Y390F (D) needed 5-, IO-, and lOO-fold, respectively, more dNTP concentration than the wild-type (A) enzyme to incorporate the first nucleotide. The relative increase in dNTP concentration needed to further elongate the primer was the same in all the mutants; thus, when the dNTP concentration was 300-fold greater than the one needed to incorporate the first nucleotide (300 nM for wild-type, 1.5 pM for mutant Y254F, and 3 p M for mutant Y390S) the polymerases replicated the primed oligonucleotide. The band corresponding to the fully replicated oligonucleotide in the case of mutant Y390F was not detected because the highest dNTP concentration assayed was 10 p~, and mutant protein Y390F would need 30 pM to replicate the oligonucleotide. All these results indicate that mutants Y254F, Y390S, and, especially, Y390F have lower affinity for dNTPs than wild-type 429 DNA polymerase, but they do not seem to be affected in translocation. Furthermore, taking into account that the three mutants have normal levels of 3' + 5' exonuclease activity (Blanco et al., 1991; see below), the lower affinity for Mg2+-dNTPs in these mutants unbalances the dynamic equilibrium between synthesis and degradation, and higher dNTP concentration is required to undergo the first polymerization event.
Mutant Proteins in Residue Ty?" of 429 DNA Polymerase Are Hypersensitive to the dNTP Analogs BuAdATP and BuPdGTP Wild-type 429 DNA polymerase has been shown to be sensitive to the dNTP analogs BuAdATP and BuPdGTP (Blanco and Salas, 1986;Bernad et al., 1987), not only in the synthetic activities (protein-primed initiation and DNA polymerization) but also in the 3' * 5' exonuclease activity.
Sensitivity to these drugs has been one of the criteria to include 429 DNA polymerase in the group of a-like DNA polymerases, although the sensitivity of 429 DNA polymerase to these drugs is lower than in the case of other a-like DNA polymerases. Moreover, in 429 DNA polymerase this sensitivity is not based on template complementarity (Blanco and Salas, 1986), contrary to other a-like DNA polymerases .
The sensitivity of wild-type and mutant 429 DNA polymerases to BuAdATP and BuPdGTP when assayed for different synthetic activities is shown in Fig. 6. The drug concentrations at which the Mn2+-dependent control activities of wild-type and mutant $29 DNA polymerases are reduced to 50% are defined as IC,, values. In processive replication assays, mutant proteins Y390F and Y390S were hypersensitive, relative to the wild-type enzyme, to BuAdATP (Fig. 6A) and BuPdGTP (not shown), the IC5, values for BuAdATP being 0.2 and 0.4 p~, respectively, in the case of TPsDNA replication and 0.1 and 0.3 p~, respectively, in the case of primed-M13 DNA replication. Mutant Y254F 429 DNA polymerase was not hypersensitive to the drugs, the IC,, values for BuAdATP being 15 p~ in TP .DNA replication and 38 p~ in primed-M13 DNA replication, similar to the ones for wild-type 429 DNA polymerase (20 p~ in TP-DNA replication and 17 p~ in primed-M13 DNA replication).
Contribution of template complementarity to the sensitivity to BuAdATP and BuPdGTP of wild-type and mutant 429 DNA polymerases was studied in filling-in polymerization assays (see "Materials and Methods"). As shown in Fig. 6B, when EcoRI-digested DNA was used as template for the filling-in reaction in the presence of [a-32P]dATP, it was found that mutants in residue Tyr3'0 were hypersensitive to BuAdATP (in this case the nucleotide analog is complementary to the two first positions of the template and competes with the insertion of labeled dATP), the ICs0 values being 12 and 3 p~, respectively, for mutants Y390S and Y390F; the ICs0 values for wild-type and mutant Y254F were 42 and 24 pM, respectively. On the other hand, no hypersensitivity to BuPdGTP (non-complementary to the template nucleotides) of mutant proteins in residue T y P was found with the latter template, the ICbo values for wild-type and mutant 429 DNA polymerases being about 30 p~ (not shown). However, when BamHI-digested DNA was used as template for the filling-in reaction in the presence of [cP~'P]~GTP, it was found that mutants in residue Tyr3" were hypersensitive to BuPdGTP filling-in reaction using either EcoRI-digested DNA or BamHI-digested DNA as templates. The reaction conditions were as described under "Materials and Methods"; in filling-in reaction using Eco RIdigested DNA as template the ICSo values for BuAdATP were 42, 24, 12, and 3 PM for wild-type and mutant Y254F, Y390.3, Y390F $29 DNA polymerases, respectively; in filling-in reaction using BamHIdigested DNA as template the IC, values for BuPdGTP were 20, 24, 11, and 4 p~ for wild-type and mutant Y254F, Y390S, Y390F $29 DNA polymerases, respectively. C, TP. DNA-dependent initiation assay. The reaction conditions were as described under "Materials and Methods," in the presence of 20 ng of TP and 20 ng of wild-type or mutant $29 DNA polymerases. Incubation was for 2 min at 30 "C; the IC50 values for BuPdGTP were 7 ~L M for wild-type and mutant $29 DNA polymerases; the ICs0 values for BuAdATP were 39, 26, 1, and 11 p~ for wild-type and mutant Y254F, Y390S, and Y390F $29 DNA polymerases, respectively. The activity in the absence of drugs was considered 100% in all cases (control activity).
(the nucleotide analog is complementary to the first template nucleotide and competes with the insertion of labeled dGTP), the IC,, values being 11 and 4 p~, respectively, for Y390S and Y390F; the ICbo values for wild-type and mutant Y254F DNA polymerases were 20 and 35 p~, respectively (see Fig.  6B). On the other hand, when the dNTP analog used in this filling-in reaction was BuAdATP (not competing by complementarity with labeled dGTP insertion), no hypersensitivity was found in mutants in residue Tyr"', the IC,, values for wild-type and mutant 429 DNA polymerases being about 50 VM (not shown). Therefore, the hypersensitivity of mutants Y390F and Y390S to the dNTP analogs is template-dependent. In agreement with this template-dependent hypersensitivity, when mutants in Tyr", were assayed in the proteinprimed initiation step of 429 DNA replication, which consists in the template-directed formation of TP. dAMP, the reaction was hypersensitive to BuAdATP, but not to BuPdGTP (Fig.  6C); the ICs0 values for wild-type and mutant 429 DNA polymerases in the case of BuPdGTP were about 7 p~, whereas those for BuAdATP were 1 and 11 VM, respectively, for mutants Y390S and Y390F and 39 and 26 VM, respectively, for wild-type and mutant Y254F DNA polymerases. Although both mutants, Y390S and Y390F, are hypersensitive to these nucleotide analogs, mutant Y390F showed the strongest phenotype when assayed in polymerization activity, whereas mutant Y390S was the most hypersensitive one in TP .DNA initiation, suggesting significant differences in the binding site when either DNA or a protein is used as primer. Furthermore, when no template was used in the formation of the TP. dAMP complex (see Blanco et al., 1992; this paper) no hypersensitivity either to BuAdATP or to BuPdGTP was found in any of the mutant proteins, their ICso values for wild-type and mutant 429 DNA polymerases being about 20 VM (not shown).
3' + 5' Exonuclease Activity of Mutants Y254F, Y390S, and Y390F The 3' + 5' exonuclease activity of mutants Y254F, Y390S and Y390F with respect to that of wild-type 429 DNA polymerase using Mg2+ as activator is shown in Fig. 7. As expected from the N-terminal location proposed for the 3' + 5' exonuclease domain (Bernad et al., 1989), mutant DNA polymerases had no reduced exonuclease activity on ssor dsDNA, the activity being about 2-fold higher than that of wild-type 429 DNA polymerase. Similar results were obtained when Mn2+ was used as metal .activator (not shown).
The 3' + 5' exonuclease activity of $29 DNA polymerase has been shown to be sensitive to BuAdATP and BuPdGTP (Blanco and Salas, 1986). Taking into account that this activity is located in the N-terminal domain of the 429 DNA polymerase, and that synthetic activities are located in the C-

~3 w r
", Y 2 U l >3WIS Y3WI nc: 5 10 2.5 5 2.5 5 2.5 5 5 10 2.5 5 2.5 5 2.5 5 " " " " I . 3' 4 5' exonuclease activity on ss-and dsDNA of wild-type or mutant $29 DNA polymerases. The reaction conditions were as indicated under "Materials and Methods" in the presence of the indicated amount of either wild-type or mutant 629 DNA polymerases. A, 5"labeled SP1 was used as ssDNA substrate for the 3' + 5' exonuclease activity of either wild-type or mutant 629 DNA polymerases. B, hybrid between 5'4abeled SP1 and SPlc+6 was used as dsDNA substrate. terminal domain, it was interesting to test if mutants that are hypersensitive to BuAdATP and BuPdGTP (mutants in Tyr"') have a 3' * 5' exonuclease activity with normal sensitivity to the drugs. As expected, mutations in residue Tyr""" did not affect the sensitivity of the 3' "-* 5' exonuclease activity to BuAdATP and BuPdGTP (results not shown).

DISCUSSION
429 DNA polymerase mutants in residues TyrZs4 (Y254F) and Tyr"" (Y390S and Y390F), localized in two regions conserved among a-like DNA polymerases, and characterized by the amino acids motifs D-NSLYP and K-NS(L/V)YG, respectively, are affected in polymerase synthetic activities (initiation and elongation), but not in 3' + 5' exonuclease activity (Blanco et al., 1991;this paper). Tyr2j4 and TyrS9" of $29 DNA polymerase are among the most conserved residues in each of the two motifs D-NSLYP and K-NS(L/V)YG of a-like DNA polymerases; in addition, these two motifs show significant amino acid cross-similarity (the consensus between them being "NSLY"). Furthermore, several mutations conferring altered sensitivity to dNTP analogs or to drugs that affect dNTP binding have been described to map in/or near these two conserved motifs (Larder et al., 1987;Gibbs et al., 1988;Matthews et al., 1989;Hall et al., 1989;De Filippes, 1989;Matsumoto e t al., 1990), suggesting the possibility that they may play an important role in the active site of cu-like DNA polymerases, most likely affecting dNTP binding.
429 DNA polymerase mutant Y254F was defective in TP. DNA dependent initiation due to a reduced affinity for Me2+-dATP, the initiating nucleotide. The K,,, value for Mg2+-dATP was 15-fold higher in mutant Y254F than in wild-type 429 DNA polymerase and 4-fold higher for Mn2+-dATP. In addition, in the non-templated deoxynucleotidylation of TP, 429 DNA polymerase mutant Y254F showed a different pattern of dNTP usage than the wild-type enzyme. These data suggest that Y254F mutant has an altered dNTP binding site.
The polymerization activity (M13 DNA or TP.DNA replication) of mutants in residues Tyr254 and Tyr3" was reduced compared to wild-type 429 DNA polymerase. The defective polymerization activity of these mutants could be overcome using Mn" instead of M$+ as metal activator. The fact that the ability for processive replication of the mutant polymerases is strongly dependent on the nature of the quelate Me'+-dNTP suggests that both residues, T Y~"~ and Tyr"", directly contribute to the Me'+-dNTP binding. A similar effect was also observed in the filling-in reaction, being all the mutants as active as the wild-type enzyme in the labeling of EcoRI ends when MnP+ was used as metal activator. Moreover, the high exonuclease activity coupled to the filling-in reaction (turnover) that was observed for mutants Y254F, Y390S, and Y390F when Mg'C was used as metal activator (62-fold for Y254F, 22-fold for Y390S, and 16-fold for Y390F, according to Blanco et al. (1991)) became similar to that of the wildtype enzyme when Mn2+ ions were used. Taking into account that in the absence of dNTPs the 3' + 5' exonuclease activity of mutants Y254F, Y390S, and Y390F was only about 2-fold higher than that of wild-type 429 DNA polymerase, and similar with either Mg2+ or Mn2+ ions, the high turnover of mutants Y254F, Y390S, and Y390F observed in the presence of Mg'+ ions probably reflects an increase in the internal proofreading described by Eger et al. (1991), which occurs when the 3' terminus is still at the nucleotide binding site after the polymerization event. A slight but important modification of the dNTP binding site in the case of mutants Y254F, Y390S, and Y390F could make this situation more similar to the case of a mismatch insertion, in which the equilibrium between synthesis and degradation is altered in favor of the exonuclease activity. On the other hand, in the presence of Mn2+, the internal proofreading of the mutants would be equivalent to that of wild-type 429 DNA polymerase allowing normal processive TP-DNA and primed-M13 DNA replication.
In 429 DNA polymerase, the equilibrium between synthesis (DNA polymerization) and degradation (3' -P 5' exonuclease) is dependent on the dNTP concentration used. Because all the mutant $29 DNA polymerases had a similar exonuclease activity as the wild-type enzyme, the requirement of higher dNTP concentration for polymerization by mutant 429 DNA polymerases relative to the wild-type enzyme (100 times higher in the case of mutant Y390F) suggests that in mutants at positions Tyr254 and Tyr390 the affinity or stability of the dNTP in the active site of the protein is lower than in wildtype 429 DNA polymerase. The results on the higher resistance to ddATP of mutant 429 DNA polymera~es,~ especially mutant Y390F, relative to the wild-type enzyme, also support this idea.
The nucleotide analogs BuAdATP and BuPdGTP have been described as potent and highly selective inhibitors of mammalian DNA polymerase a . $29 DNA polymerase and other a-like DNA polymerases have been described to be sensitive to these nucleotide analogs, that can be considered as specific probes to study the dNTP binding site. BuAdATP and BuPdGTP have an aryl domain and a base-pairing domain; the first one is proposed to interact with the dNTP binding site of the polymerase and the second one to interact with the complementary base in the template . In calf thymus DNA polymerase a, the inhibitory effect of these drugs is strongly enhanced when they are complementary to the template nucleotide . $29 DNA polymerase has been described as sensitive to BuAdATP and BuPdGTP in protein-primed initiation of TP.DNA replication, DNA polymerization and 3' -P 5' exonuclease reactions (Blanco and Salas, 1986). However, there are clear differences with respect to mammalian DNA polymerase a; in 429 DNA polymerase, inhibition of the initiation reaction (TP-dAMP complex formation) occurs with both BuAdATP and BuPdGTP, indicating non-template dependence, and the affinity of 429 DNA polymerase for these drugs is lower than the one of mammalian DNA polymerase a (not shown ing that Tyr3" is an important residue in template-dependent dNTP binding. The Klenow fragment of pol I, whose crystal structure is known (Ollis et al., 1985), is the prototype of a group of DNA polymerases (pol I group), including bacterial, viral, and mitochondrial enzymes (reviewed by Blanco et al. (1991) and Ito and Braithwaite (1991)). Because the three-dimensional structure of none of the a-like DNA polymerases is known, primary structure alignments between a-like and pol I-like DNA polymerases have been obtained, to locate, on the Klenow crystal, the most conserved amino acid motifs (Matsu-mot0 et al., 1989;Delarue et al., 1990;Blanco et al., 1991). However, the alignments reported differ considerably, probably due to the existence of structural, and perhaps functional, duplication of several conserved regions. Thus, three different invariant tyrosine residues in a-like DNA polymerases were considered to be homologous to Tyr766 of pol I, directly involved in dNTP binding (Rush and Konigsberg, 1990): the tyrosine contained in the D-NSLYP motif (Matsumoto et al., 1989), the tyrosine contained in the K-NS(L/V)YG motif (Delarue et al., 1990), and the tyrosine corresponding to the "K-Y" consensus in region 4 (Blanco et ai, 1991). Therefore, the D-NSLYP motif has been extrapolated to a-helix 0 (Matsumoto et al., 1989) and the K-NS(L/V)YG motif has been extrapolated either to a-helix I (Blanco et al., 1991) or to a-helix 0 (Delarue et al., 1990), in pol I three-dimensional structure. Interestingly, although a-helix I has been mainly involved in DNA binding (Basu et al., 1988), and a-helix 0 in dNTP binding (Joyce and Steitz, 1987), there is a considerable number of similarities among these two regions of the pol I structure. 1) Both regions have a similar three-dimensional location (forming opposite walls of the pol I cleft). 2) Both have a similar secondary structure (a-helix) and are flanked by flexible and disordered amino acid spans. 3) Both contain a lysine residue (invariant in pol I group) located in a central position (see Blanco et al., 1991); in pol I, these two lysines (K635 and K758) react with pyridoxal phosphate (Basu et al., 1988;Basu and Modak, 1987), and they are located diagonally with respect to the DNA binding cleft. 4) Both regions have a tyrosine residue highly conserved or invariant in the pol I group located in a similar relative position with respect to the invariant lysine (Blanco et al., 1991). These structural and perhaps functional similarities, together with the cross-similarity of the regions containing the D-NSLYP and K-NS(L/ V)YG) motifs of a-like DNA polymerases, could explain the different alignments reported (Matsumoto et al., 1989;Delarue et al., 1990;Blanco et al., 1991).
According to the results presented in this paper, residues T y P and of conserved motifs D-NSLYP and K-NS(L/V)YG, respectively, seem to be involved, directly or indirectly, in dNTP binding by $29 DNA polymerase; mutation TyrZ54 to Phe affects dNTP binding, mainly in the formation of TP.dNMP complex when TP is used as primer, and also affects the affinity for Mg2'-dNTP quelate and its stability at the active site in polymerization assays. Mutations Tyr3" to Ser or to Phe also affect 429 DNA polymerase affinity for Mg2+-dNTP quelate and its stability at the active site in polymerization assays, but do not affect dNTP binding when TP is used as primer. This fact suggests significant differences in the dNTP binding site when either DNA or a protein is used as primer. Moreover, mutations in Tyr3'0 also affect the $29 DNA polymerase affinity for dNTP analogs in a way that is template-dependent. It is likely that more residues, not necessarily close in the primary structure, are also relevant for a proper dNTP binding, defining a region in a-like DNA polymerases similar to the polymerase active site dNTP Binding of pol I Klenow fragment (Polesky et al., 1990).