©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Primer Terminus Stabilization at the 29 DNA Polymerase Active Site
MUTATIONAL ANALYSIS OF CONSERVED MOTIF KXY (*)

(Received for publication, August 5, 1994; and in revised form, November 28, 1994)

María A. Blasco(§)(¶) Juan Méndez (§) José M. Lázaro Luis Blanco Margarita Salas (**)

From the Centro de Biología Molecular ``Severo Ochoa'' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. A conserved amino acid motif, located in the C-terminal portion of the polypeptide and characterized by the amino acid sequence KK(K/R)Y, is conserved in the group of eukaryotic-type DNA polymerases. In the subgroup of DNA polymerases that have a protein-priming mechanism, this motif is restricted to the sequence KXY, X never being a positively charged amino acid. Residues Lys and Tyr form this conserved motif in 29 DNA polymerase. Mutant K498T, in which the positive charge of the motif has been eliminated, was strongly affected both in initiation (terminal protein-dAMP formation, using terminal protein as primer) and DNA polymerization reactions. Mutants K498R and Y500S were able to carry out the initiation reaction to a higher or similar extent, respectively, than wild-type 29 DNA polymerase but were affected in DNA polymerization reactions. All of the mutations severely affected the stable binding of the polymerase to a primer-template DNA. In addition, all of the mutant polymerases analyzed in this work showed an unusually strong 3`-5` exonuclease activity both under polymerization or non-polymerization conditions. The results obtained suggest a role of the conserved residues of the KXY motif in stabilizing the primer terminus at the polymerization active site, the positive charge of residue Lys being critical for the synthetic activities of 29 DNA polymerase.


INTRODUCTION

The 29 genome is a linear, double-stranded DNA, with a TP (^1)covalently linked to both 5`-ends. This linear genome solves the problem of complete replication of the DNA ends by a protein-primed initiation mechanism (for a review, see Salas, 1991). To initiate TP-DNA replication, 29 DNA polymerase is able to use the 3`-OH group of residue Ser of a free TP as primer, catalyzing the formation of TPbulletdAMP, the initiation complex (Blanco and Salas, 1984; Hermoso et al., 1985). This initiation complex is further elongated by 29 DNA polymerase in a process that requires high processivity and strand-displacement ability, two properties of this enzyme (Blanco and Salas, 1985a). In addition, 29 DNA polymerase has two degradative activities, a 3`-5` exonuclease activity (Watabe et al., 1984; Blanco and Salas, 1985b) involved in proofreading (Garmendia et al., 1992) and a pyrophosphorolytic activity (Blasco et al., 1991).

29 DNA polymerase consists of a single polypeptide (66 kDa), and its enzymatic activities (protein-primed initiation, DNA polymerization, 3`-5` exonuclease, and pyrophosphorolysis) have been well characterized. Therefore, it is an appropriate system for studies to relate structure and function. 29 DNA polymerase has been included in the group of eukaryotic-type DNA polymerases because of its sensitivity to drugs that are specific inhibitors of eukaryotic DNA polymerase alpha (Blanco and Salas, 1986; Bernad et al., 1987) and because it shares with this group of polymerases several regions of amino acid similarity along the primary structure (Blanco et al., 1991).

In addition to the evolutionary conserved 3`-5` exonuclease active site, present in the N-terminal domain of both prokaryotic- and eukaryotic-type DNA polymerases (Bernad et al., 1989; reviewed by Joyce and Steitz, 1994), several amino acid sequence motifs conserved in the C-terminal portion of eukaryotic-type DNA polymerases have been proposed to be part of the active site for synthetic activities (Larder et al., 1987; Gibbs et al., 1988). The functional significance of the conserved motifs DX(2)SLYP, KX(3)NSXYG, TX(2)GR, and YXDTDS has been previously studied by site-directed mutagenesis in 29 DNA polymerase; most of the mutant polymerases showed an essentially normal 3`-5` exonuclease activity, but they were affected in synthetic activities (protein-primed initiation and/or polymerization) and in pyrophosphorolysis. Thus, 29 residues Asp (in conserved motif DX(2)SLYP) and Asp and Asp (in conserved motif YXDTDS) have been proposed to be involved in catalysis, probably contributing to metal binding (Bernad et al., 1990; Blasco et al., 1993a). Conserved motif KX(3)NSXYG has been proposed to be involved in primer-template binding and dNTP selection (Blasco et al., 1992a, 1993b). Thr and Arg, belonging to conserved motif TX(2)GR, have been proposed to be involved in primer binding (Méndez et al., 1994). In addition, a mutant in residue Tyr of 29 DNA polymerase conserved motif DX(2)SLYP has been described to be affected in dNTP binding (Blasco et al., 1992a).

Other amino acid motifs in the C-terminal portion of eukaryotic-type DNA polymerases have been also described (Blanco et al., 1991; Braithwaite and Ito, 1993); conserved amino acid motif KK(K/R)Y is found in most eukaryotic-type DNA polymerases described up to now, with the exception of those involved in a protein-priming mechanism. In the latter, this motif is restricted to KXY. Preliminary data with crude extracts have indicated the importance of 29 DNA polymerase motif KXY in synthetic activities (Blasco et al., 1992b). In this paper, a detailed characterization of purified 29 DNA polymerase mutants K498R, K498T, and Y500S allows us to conclude that 29 DNA polymerase conserved motif KXY forms part of the active site for synthetic activities, probably contributing to primer terminus stabilization.


MATERIALS AND METHODS

Proteins and DNA

Wild-type or 29 DNA polymerase mutants were purified essentially as described (Lázaro et al., 1994). TP was purified as described (Zaballos et al., 1989). TP-DNA was isolated as described (Peñalva and Salas, 1982). Oligonucleotides sp1 (5`-GATCACAGTGAGTAC), sp1c (5`-GTACTCACTGTGATC), and sp1c+6 (5`-TCTATTGTACTCACTGTGATC) were prepared, purified, and labeled with polynucleotide kinase and [-P]ATP, as described (Garmendia et al. 1992). Hybrid molecules sp1/sp1c (15/15-mer), sp1/sp1c+6 (15/21-mer), and primed M13 mp2 DNA were obtained as described (Garmendia et al., 1992). EcoRI-digested 29 DNA was prepared from proteinase K-treated 29 DNA (Inciarte et al., 1976). P-dA-tailed DNA, prepared by extending PstI-digested pUC19 with terminal deoxynucleotidyl transferase and [alpha-P]dATP, was used as substrate for quantitative analysis of the 3`-5` exonuclease activity. As substrate for pyrophosphorolytic activity, EcoRI-digested 29 DNA was 3`-labeled with [alpha-P]dATP in a partial filling-in reaction carried out by an exonuclease-deficient mutant 29 DNA polymerase (D12A/D66A; Bernad et al., 1989).

Plasmids and Bacteria

Plasmid pMBw2, harboring the 29 DNA polymerase gene, was obtained as described (Blasco et al., 1990). The Escherichia coli strain K514 was used as a host for transformation with pT7-4 (Tabor and Richardson, 1985) recombinants containing the 29 DNA polymerase gene.

Site-directed Mutagenesis and Expression of 29 DNA Polymerase Mutants

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 (Nakamaye and Eckstein, 1986). For expression, fragments carrying the different mutations were cloned in plasmid pMBw2 (not shown). The presence of the desired mutations and the absence of any other changes was confirmed by complete sequencing of each 29 DNA polymerase mutant gene. Expression of the mutant proteins was carried out in the E. coli strain BL21(DE3) pLysS (Studier and Moffatt, 1986).

3`-5` Exonuclease Assays

For quantitative analysis, P-dA-tailed DNA (PstI-digested pUC19; 3.3 times 10^6 cpm/pmol of 3`-end) was used as substrate of the exonucleolytic activity of wild-type or mutant 29 DNA polymerases, as described (Méndez et al., 1994). When indicated, the dAMP turnover coupled to filling-in DNA polymerization assays (see later) was determined as described (Méndez et al., 1994).

DNA Polymerization Assays

Analysis of DNA polymerization activity by filling-in of 3`-recessive DNA ends, primed M13 DNA replication, or polymerase/exonuclease-coupled assays, was carried out as described (Méndez et al., 1994).

Gel Retardation Assay of Primer-Template DNA Molecules

The hybrid oligonucleotide 5`-P-sp1/sp1c+6 (15/21-mer) was used as primer template for analysis of its interaction either with wild-type or mutant 29 DNA polymerase. Protein-DNA complexes were analyzed by gel retardation assays, performed as described by Méndez et al. (1994), and detected as a shift (retardation) in the position of the labeled DNA.

TPbulletdAMP Formation (Initiation Assay)

When TP-DNA was used as template, the reaction mixture contained, in 25 µl, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl(2), 20 mM ammonium sulfate, 0.1 mg/ml bovine serum albumin, 1 mM dithiothreitol, 4% glycerol, 50 mM NaCl, 0.25 µM [alpha-P]dATP (2.5 µCi), 20 ng of purified TP, 20 ng of either wild-type or mutant DNA polymerase, and TP-DNA (0.5 µg) as template. Incubation was at 30 °C during 5 min. To study template-independent TP deoxynucleotidylation, reaction conditions were as described above, using 1 mM MnCl(2) as metal activator instead of MgCl(2), in the absence of TP-DNA and in the presence of 100 ng of purified TP and either wild-type or mutant 29 DNA polymerase; incubation was for 4 h at 25 °C. In all cases, after incubation, 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 SDS-polyacrylamide gel electrophoresis and autoradiography. Quantitation was done by densitometry of the radioactive bands corresponding to TPbulletdAMP.

Competition Assay for TP Binding between Wild-type and Mutant 29 DNA Polymerases

Reactions were carried out as described for the initiation assay in the absence of TP-DNA and using invariant amounts of TP (12.5 ng) and wild-type 29 DNA polymerase (25 ng). Increasing amounts of 29 DNA polymerase mutant K498T (12.5, 25, 50, 100, and 200 ng) were added. After incubation for 3 h at 25 °C, the reactions were stopped by adding up to 10 mM EDTA, 0.1% SDS and filtered as indicated above. Quantitation was done by densitometry of the radioactive bands corresponding to TPbulletdAMP.

Elongation of TPbulletdAMP Initiation Complex

To analyze the complete elongation of TPbulletdAMP initiation complex, the incubation mixtures contained, in 25 µl, 50 mM Tris-HCl (pH 7.5), either 10 mM MgCl(2) or 1 mM MnCl(2), 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 20 mM ammonium sulfate, 20 µM of each dNTP, and 0.25 µM [alpha-P]dATP (2.5 µCi), 0.5 µg of TP-DNA, 20 ng of TP, and 20 ng of either wild-type or mutant 29 DNA polymerase. After incubation for 10 min at 30 °C, the reactions were stopped by adding up to 10 mM EDTA, 0.1% SDS and filtered as indicated above. Samples were subjected to alkaline 0.7% agarose gel electrophoresis and autoradiography. Quantitation was done by densitometry of the radioactive bands.

To study the truncated elongation of TPbulletdAMP, the final dATP concentration was varied by adding different concentrations of unlabeled dATP and keeping constant the [alpha-P]dATP (2.5 µCi); unlabeled dGTP and dTTP were also added at 10 µM final concentration. The reaction was performed in the presence of Mn as metal activator. For the analysis of the truncated elongation products, electrophoresis was carried out in 0.1% SDS, 12% polyacrylamide gels (360 times 280 times 0.5 mm), conditions in which the different TPbullet(dNMP)(n) complexes are resolved.

Pyrophosphorolysis

Wild-type or mutant 29 DNA polymerase were assayed for pyrophosphorolysis essentially as described by Blasco et al.(1991). Aliquots of each reaction mixture were subjected to thin layer chromatography in polygram Cel 300 PEI/UV254 and developed in 0.5 M potassium phosphate buffer. After drying, the plates were subjected to autoradiography. Quantitation was done by densitometry of the spot corresponding to 5`-[P]dATP.


RESULTS

Site-directed Mutagenesis in 29 DNA Polymerase-conserved Motif KXY

A conserved motif characterized by the sequence KK(K/R)Y is present in most eukaryotic-type DNA polymerases (Blanco et al., 1991; Braithwaite and Ito, 1993). In Fig. 1, an updated multiple alignment of this region is presented. According to this new alignment, two of the sequences corresponding to the group of bacterial/viral DNA polymerases considered in Fig. 1present some differences with the consensus KK(K/R)Y. Thus, channel catfish virus DNA polymerase has the first lysine and the tyrosine changed into leucine, and Sulfolobus solfataricus DNA polymerase has the third position in the motif (K/R) changed into asparagine. In the group of cellular enzymes, all of the alpha DNA polymerases have the motif KKKY, whereas in , , and Rev3 DNA polymerases, the sequence of the motif is always KKRY. In the group of polymerases that use a protein-priming mechanism, the sequence of the conserved motif has two significant differences: (a) the first Lys residue of the motif is not conserved and (b) the third residue is always a non-positively charged amino acid. Therefore, KXY would be a more strict consensus for all the eukaryotic-type DNA polymerases (see Fig. 1). Residues Lys, Thr (not conserved), and Tyr form the KXY motif in 29 DNA polymerase. Mutants K498T, K498R, T499A, and Y500S of this motif have been previously studied using crude fractions (Blasco et al., 1992b). In this paper, mutants K498R, K498T, and Y500S were selected for a detailed biochemical characterization. These mutant polymerases were overexpressed and purified as described (see ``Materials and Methods'').


Figure 1: Sequence alignment in eukaryotic-type DNA polymerases for conserved amino acid motif characterized by the sequence KXY. The amino acids belonging to the conserved motif KK(K/R)Y (in the groups of bacterial, viral, and cellular DNA polymerases) or KXY (DNA polymerases that initiate by protein priming) are indicated in whiteletters over a blackbackground. Other relevant amino acid similarities among the different groups of DNA polymerases are boldfacetype in grayboxes. The following conservative amino acids were considered: S and T; A and G; K, R, and H; D, E, Q, and N; I, L, C, M, V, Y, and F. DNA polymerase nomenclature and references are compiled in Braithwaite and Ito(1993), with the following exceptions: African swine fever virus DNA polymerase (ASFV) (Rodríguez et al., 1993), DNA polymerase from mouse ( (Mm)) (Cullmann et al., 1993), DNA polymerase from Saccharomyces cerevisiae ( (Sc)) (Morrison et al., 1989), DNA polymerase encoded by a linear plasmid from Morchella conica (pMC3-2; Rohe et al., 1991), and DNA polymerases from Chlorella viruses, strains NY-2A (SwissProt data base, accession number P30320) and PBCV-1 (SwissProt data base, accession number P30321). Numbers between slashes indicate the amino acid position relative to the N-terminus of each DNA polymerase. The numbers in parentheses indicate insertions of 8 amino acids.



Degradative Activities by Wild-type and Mutant 29 DNA Polymerases

3`-5` Exonuclease Activity

The 3`-5` exonuclease activity of the different mutant DNA polymerases was measured as degradation of P-dA-tailed DNA (see ``Materials and Methods''). As shown in Table 1, single substitutions of conserved residues Lys and Tyr produced an increase in the 3`-5` exonuclease activity that was 3-4-fold higher than that of the wild-type polymerase. A possible explanation of this strong 3`-5` exonuclease activity will be discussed later.



Pyrophosphorolysis

Pyrophosphorolysis, the reversal reaction of DNA polymerization, occurs only in the presence of high concentrations of PPi, and it is catalyzed at the polymerization active site (Blasco et al., 1990). Mutants K498R, K498T, and Y500S resulted severely affected in this reaction (see Table 1).

DNA Polymerization Activity of Wild-type and Mutant 29 DNA Polymerases

Wild-type and mutant 29 DNA polymerases were tested in filling-in of EcoRI ends, a polymerization assay in which processivity is not critical. In the presence of Mg as metal activator, mutants K498R, K498T, and Y500S showed 8, 1, and 4%, respectively, wild-type activity (see Table 1). When Mn was used, mutants K498R, K498T, and Y500S were 61, 3, and 50% active, respectively (see Table 1and Fig. 2A). The [P]dAMP released by the 3`-5` exonuclease after polymerization (turnover) was also determined. The results obtained indicate that, in the presence of Mg, the turnover was 6-, 7-, and 3-fold higher in the case of mutants K498R, K498T, and Y500S, respectively, than in the case of wild-type 29 DNA polymerase (Table 1). A similar turnover was obtained when Mn was used as metal activator ( Table 1and Fig. 2B).


Figure 2: Effect of Mn in the filling-in reaction and coupled turnover of wild-type (WT) and mutant 29 DNA polymerases. A, filling-in of EcoRI-protruding ends. Reaction conditions were as described by Méndez et al.(1994), using 1 mM MnCl(2). B, 3`-5` exonuclease activity coupled to the filling-in assay (turnover), analyzed as described by Méndez et al.(1994). ori, origin of the chromatogram.



The ability of the different mutant polymerases to carry out processive DNA polymerization was analyzed in a primed M13 DNA replication assay (see ``Materials and Methods''). When Mg was used as metal activator, mutants K498R, K498T, and Y500S showed essentially no activity (see Table 1); when Mn was used, mutant polymerases K498R and Y500S partially recovered activity.

To study more precisely the balance between polymerase and exonuclease activities, mutants K498R, K498T, and Y500S were analyzed in a polymerase/exonuclease assay (see ``Materials and Methods''). In this assay, the elongation of a 5`-labeled primer (15-mer) hybridized to a short template molecule (21-mer) was analyzed in the presence of Mg as metal activator. When increasing amounts of unlabeled dNTPs are provided, elongation competes exonucleolysis, and dNTP incorporation is observed as an increase in the size of the labeled primer. Wild-type 29 DNA polymerase needed 0.05 µM dNTPs to compete out its 3`-5` exonuclease activity ( Fig. 3and Table 1). Mutant K498R needed 10 µM dNTPs to compete out its 3`-5` exonuclease activity ( Fig. 3and Table 1) and mutant K498T was unable to compete out its exonuclease activity when dNTP concentrations up to 500 µM were added (see Table 1). Mutant Y500S was able to compete out its 3`-5` exonuclease activity with 1 µM dNTPs (see Table 1). Interestingly, in the absence of dNTPs (see Fig. 3for mutant K498R), mutants K498R, K498T, and Y500S showed a stronger exonuclease activity as compared with the wild-type polymerase. This strong exonuclease on primer-template structures could be reflecting the instability of the primer terminus at the polymerization active site, being then favored its degradation by the 3`-5` exonuclease activity instead of its processive elongation. This hypothesis is also supported by the increased dNMP turnover coupled to DNA polymerization displayed by all of these mutant polymerases (see above).


Figure 3: DNA polymerase/exonuclease-coupled assay. The assay was carried out as described by Méndez et al.(1994), using P-labeled hybrid molecules SP1/SP1c+6 as primer-template DNA, the indicated concentration of each dNTP (nM), and 10 ng of wild-type (WT) or mutant K498R DNA polymerases. Arrows indicate sizes of primer strand, from 15-mer (non-elongated primer) to 21-mer (completely elongated primer).



Affinity of Mutant 29 DNA Polymerases for Primer-Template Structures (Gel Retardation Assays)

The affinity of wild-type and mutant 29 DNA polymerases for primer-template DNA molecules was studied using gel retardation assays. Wild-type 29 DNA polymerase is able to retard labeled hybrid 15/21-mer molecules, giving rise to a single retardation band; this stable interaction is interpreted to correspond to an enzyme-DNA complex competent for polymerization (Méndez et al., 1994). As shown in Fig. 4, the three mutant DNA polymerases showed a decreased capacity to produce this retardation band, in particular, in the case of mutant K498T. Interestingly, the three mutant DNA polymerases gave rise to a band (below the one corresponding to free 15/21-mer DNA) (see Fig. 4) that is not present in the case of wild-type DNA polymerase. The DNA in this band was extracted from the gel and further analyzed by electrophoresis in a 8 M urea, 20% acrylamide sequencing gel, revealing that it corresponds to small DNA fragments, 3-8 nucleotides long (results not shown). These DNA fragments are probably the consequence of the strong 3`-5` exonuclease activity on primer-template DNA molecules displayed by these mutant polymerases as a consequence of a poor or null stabilization at the polymerization active site.


Figure 4: Interaction of the wild-type (WT) and mutant DNA polymerases with a primer-template DNA. A labeled hybrid molecule of two oligonucleotides (15/21-mer) was incubated either with the wild-type or with the indicated mutant DNA polymerase. Samples were analyzed by gel electrophoresis in the conditions described by Méndez et al.(1994). The bands corresponding to free DNA and to the DNA polymerase-DNA complex are marked. In the case of the mutant DNA polymerases, the band detected below free DNA (marked with an arrow) was determined to correspond to small fragments of labeled DNA, probably produced by exonucleolytic degradation of the 15-mer (see text).



TP-primed Synthetic Activities

TPbulletdAMP Formation (Initiation of 29 DNA Replication)

The ability of the different mutant polymerases to carry out TPbulletdAMP formation, a template-directed reaction in which the DNA polymerase has to use a free molecule of TP as primer, was studied. In the presence of Mg as metal activator, mutant K498R showed 620% the wild-type initiation activity (see Table 2). The hyperactivity of this mutant polymerase in the initiation reaction is due to a 2-fold decrease in the K(m) value for dATP and to a 3-fold increase in the V(max) of the reaction as compared with the wild-type enzyme (not shown). In contrast, mutant polymerase K498T showed only 2% of the wild-type activity (see Table 2), indicating that the positive charge of this residue is critical for the initiation activity using a protein as primer. Competition assays for TP between mutant K498T and the wild-type 29 DNA polymerase (see ``Materials and Methods'') showed that mutant K498T was able to inhibit TPbulletdAMP complex formation by the wild-type enzyme (not shown), ruling out a defect of this mutant polymerase in interaction with the TP. Mutant Y500S showed 60% the wild-type initiation activity in the above conditions (see Table 2). No significant differences in relative activity were obtained when Mn was used as metal activator (not shown).



TPbulletdAMP formation can be detected in the absence of template, using Mn as metal activator (Blanco et al., 1992). As shown in Table 2and in Fig. 5, the TPbulletdAMP formation by mutants K498R, K498T, and Y500S in the absence of TP-DNA was 940, 8, and 145%, respectively, that of the wild-type activity. This result suggests that the mutant phenotype obtained when the TP is used as primer is not derived from the defect of these mutants in template binding.


Figure 5: TPbulletdAMP complex formation by wild-type (WT) and mutant DNA polymerases in the absence of TP-DNA. Reaction conditions are described under ``Materials and Methods.'' The radioactive bands corresponding to TPbulletdAMP are indicated by an arrow.



Transition between Protein Priming to DNA Priming in 29 DNA Replication

In the 29 DNA replication process, there is a transition between the use of TP as primer (initiation reaction) to that of DNA as primer (polymerization). Although mutants K498R and Y500S were able to initiate to a higher or similar extent, respectively, than wild-type 29 DNA polymerase, all of the mutant polymerases were impaired in 29 DNA replication (see Table 2). Thus, it was interesting to study the transition step, carrying out a truncated elongation assay (see ``Materials and Methods''). In this assay, wild-type 29 DNA polymerase is able to elongate TPbulletdAMP initiation complex until dCTP (the nucleotide lacking in the assay) has to be incorporated, giving rise to two bands of elongated material, originated at both ends of the TP-DNA (see Fig. 6). Mutants K498R, K498T, and Y500S, although able to initiate replication, were unable to elongate the initiation complex (TPbulletdAMP) to the same extent as the wild-type 29 DNA polymerase (Fig. 6). In the case of mutant polymerase Y500S, it was possible to detect a band corresponding to TP-(dAMP)(2), the first step in which DNA is used as primer.


Figure 6: Analysis of truncated elongation products of TPbulletdAMP initiation complex by either wild-type (WT) or mutant DNA polymerases. The assay was carried out as described under ``Materials and Methods.'' The concentrations of unlabeled dATP used are indicated. In all cases, unlabeled dGTP and dTTP were present at 10 mM final concentration. TP-d(NMP)elongated products are indicated by arrows. TP-d(NMP)(8) is from the right end of 29 DNA, and TP-d(NMP)(14) is from the left end. The film corresponding to mutant polymerase K498T, whose initiation activity is very low, was overexposed to visualize the possible products of truncated elongation.




DISCUSSION

An amino acid motif, characterized by the sequence KK(K/R)Y, can be found in the C-terminal portion of many eukaryotic-type DNA polymerases. However, in the subgroup of enzymes that use a protein-priming mechanism, the first Lys residue of the motif is not conserved, and the third residue is always a non-positively charged amino acid. Therefore, KXY should be the strict consensus for all eukaryotic-type DNA polymerases. To assess the importance of this motif in the synthetic activities of the enzyme, site-directed mutagenesis was carried out in residues Lys and Tyr of 29 DNA polymerase.

Mutant K498R, in which the positive charge of the residue was increased, showed a 6-fold higher dATP incorporation using the TP as primer (TPbulletdAMP formation) with respect to the wild-type enzyme. Despite its strong dATP incorporation activity on the primer TP, mutant K498R was strongly affected in DNA polymerization reactions, both in elongation of TPbulletdAMP initiation complex and DNA polymerization on primer-template molecules. This defect in elongation can be related with the reduced ability of this mutant to stabilize primer-template molecules at the polymerization active site. Consequently, the balance between polymerase and exonuclease activities was clearly altered in favor of the last, as can be deduced from the polymerization/exonuclease-coupled assay and from the high turnover activity detected under polymerization conditions.

The importance of the positive charge at position 498 of 29 DNA polymerase was tested studying mutant K498T. This mutant polymerase resulted severely affected in both initiation and DNA polymerization activities, as well as in normal interaction with a primer template. Despite the low initiation activity of mutant K498T, in some experimental conditions it was possible to determine its strong defect in the elongation of TPbulletdAMP initiation complex formed, as it was the case for mutant K498R. Moreover, both the turnover coupled to filling-in reactions and the exonuclease activity on primer-template structures were increased, compared with the wild-type enzyme.

The role of Tyr, the other conserved residue of motif KXY, was studied by analysis of mutant Y500S. This mutant DNA polymerase was strongly affected in DNA polymerization reactions and interaction with primer-template DNA but not in the initiation reaction. Again, this mutant showed higher 3`-5` exonuclease and turnover activities (although less drastic than in the case of mutants in residue Lys).

Taking into account the results summarized above, a direct role of motif KXY of 29 DNA polymerase in catalysis, dNTP binding, or interaction with the TP can be discarded. On the contrary, a direct role of motif KXY in the stabilization of the primer terminus at the polymerization active site would explain (a) the altered polymerase/exonuclease equilibrium (favoring the latter) observed in mutants of motif KXY; (b) the absence of stable binding of these mutants to a primer-template DNA, as determined by gel retardation assays; (c) the differences observed between the use of either TP or DNA as primer (in the case of mutant polymerases K498R and Y500S) (it should be considered that whereas a DNA primer terminus is substrate of the 3`-5` exonuclease activity, the initiation complex, TPbulletdAMP, is not (Esteban et al., 1993)); and (d) the inefficient transition from TP priming to normal DNA elongation observed in all the mutant polymerases (TP-(dAMP)(2) and longer elongation products would be unstable at the polymerization active site and therefore would be further degraded by the exonuclease activity). In addition, the instability of the primer terminus at the polymerization active site, which would result in a severe impairment of translocation, would explain the inability of the polymerases mutated at the KXY motif to carry out processive DNA polymerization and pyrophosphorolysis.

On the other hand, the analysis of mutant polymerase K498T indicates that the elimination of the positive charge at this position probably produces a more drastic distortion at the active site, since no synthetic activity can be detected, even when using the TP as primer. This phenotype cannot be explained either as a consequence of the low affinity for DNA (since the initiation reaction in the absence of template DNA was also severely impaired) or as an inefficient binding to the TP (determined to be normal). Thus, although a direct role of residue Lys in catalysis cannot be ruled out, it is possible that the positive charge at position 498 of 29 DNA polymerase is critical for the correct positioning of the primer terminus (both in the DNA and in the TP) at the polymerization active site. Interestingly, another basic residue of 29 DNA polymerase (Arg), belonging to conserved motif TX(2)GR, has been recently proposed to be involved in binding both types of primer structures (Méndez et al., 1994).

It has been reported (Hwang et al., 1992) that a drug-resistant mutant of herpes simplex virus contains a single point mutation in the DNA polymerase gene, exactly in the Tyr residue of the KXY motif (Y941H). The fact that this mutation confers to the polymerase resistance to acyclovir and foscarnet led to the suggestion that this conserved motif could be forming part of the active site of the enzyme, probably contributing to dNTP binding. Actually, mutant polymerases K498R and Y500S recovered part of the DNA polymerization activities when Mn (that reduces the K(m) for dNTPs in 29 DNA polymerase) was used as metal activator. This phenotype could well be explained as a secondary effect derived from the instability of the DNA primer terminus at the active site, which would certainly impair the correct positioning and stabilization of the incoming nucleotide. However, none of the mutant polymerases studied in this work showed altered sensitivities to dNTP analogs BuAdATP and BuPdGTP, (^2)as it was the case in other 29 DNA polymerase mutants affected in dNTP binding and/or selection (Blasco et al., 1992a).

In conclusion, the phenotypes of mutants K498R, K498T, and Y500S in conserved motif KXY suggest a function of these conserved amino acids in the binding and/or stabilization of primer terminus at the polymerization active site, being the positive charge of residue Lys critical for dNTP incorporation on both TP and DNA primer terminus structures.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant 5R01 GM27242-15, by Grant PB90-0091 from Dirección General de Investigación Científica y Técnica, by Grants BIOT-CT 91-0268 and CHRX-CT93-0248 from European Economic Community, and by an Institutional grant from Fundación Ramón Areces. 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.

§
Predoctoral fellows from Ministerio de Educación y Ciencia.

Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

**
To whom correspondence should be addressed.

(^1)
The abbreviation used is: TP, 29 terminal protein.

(^2)
M. A. Blasco, J. Méndez, J. M. Lázaro, L. Blanco, and M. Salas, unpublished results.


ACKNOWLEDGEMENTS

We thank L. Villar for sequencing the complete mutant 29 DNA polymerase genes.


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  4. Blanco, L., and Salas, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 5325-5329 [Abstract]
  5. Blanco, L., and Salas, M. (1985a) Proc. Natl. Acad. Sci. U. S. A. 82, 6404-6408 [Abstract]
  6. Blanco, L., and Salas, M. (1985b) Nucleic Acids Res. 13, 1239-1249 [Abstract]
  7. Blanco, L., and Salas, M. (1986) Virology 153, 179-187 [Medline] [Order article via Infotrieve]
  8. Blanco, L., Bernad, A., Blasco, M. A., and Salas, M. (1991) Gene (Amst.) 100, 27-38 [CrossRef][Medline] [Order article via Infotrieve]
  9. Blanco, L., Bernad, A., Esteban, J. A., and Salas, M. (1992) J. Biol. Chem. 267, 1225-1230 [Abstract/Free Full Text]
  10. Blasco, M. A., Blanco, L., Parés, E., Salas, M., and Bernad, A. (1990) Nucleic Acids Res. 18, 4763-4770 [Abstract]
  11. Blasco, M. A., Bernad, A., Blanco, L., and Salas, M. (1991) J. Biol. Chem. 266, 7904-7909 [Abstract/Free Full Text]
  12. Blasco, M. A., Lázaro, J. M., Bernad, A., Blanco, L., and Salas, M. (1992a) J. Biol. Chem. 267, 19427-19434 [Abstract/Free Full Text]
  13. Blasco, M. A., Esteban, J. A., Méndez, J., Blanco, L., and Salas, M. (1992b) Chromosoma (Berl.) 102, 32-38 [Medline] [Order article via Infotrieve]
  14. Blasco, M. A., Lázaro, J. M., Blanco, L., and Salas, M. (1993a) J. Biol. Chem. 268, 24106-24113 [Abstract/Free Full Text]
  15. Blasco, M. A., Lázaro, J. M., Blanco, L., and Salas, M. (1993b) J. Biol. Chem. 268, 16763-16770 [Abstract/Free Full Text]
  16. Braithwaite, D. K., and Ito, J. (1993) Nucleic Acids Res. 21, 787-802 [Medline] [Order article via Infotrieve]
  17. Cullmann, G., Hindges, R., Berchtold, M. W., and Hübscher, U. (1993) Gene (Amst.) 134, 191-200 [CrossRef][Medline] [Order article via Infotrieve]
  18. Esteban, J. A., Salas, M., and Blanco, L. (1993) J. Biol. Chem. 268, 2719-2726 [Abstract/Free Full Text]
  19. Garmendia, C., Bernad, A., Esteban, J. A., Blanco, L., and Salas, M. (1992) J. Biol. Chem. 267, 2594-2599 [Abstract/Free Full Text]
  20. Gibbs, J., Chiou, H., Bastow, K., Cheng, Y., and Coen, D. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6672-6676 [Abstract]
  21. Hermoso, J. M., Méndez, E., Soriano, F., and Salas, M. (1985) Nucleic Acids Res. 13, 7715-7728 [Abstract]
  22. Hwang, C. B., Ruffner, K. L., and Coen, D. M. (1992) J. Virol. 66, 1774-1776 [Abstract]
  23. Inciarte, M. R., Lázaro, J. M., Salas, M., and Viñuela, E. (1976) Virology 74, 314-323 [Medline] [Order article via Infotrieve]
  24. Joyce, C. M., and Steitz, T. A. (1994) Annu. Rev. Biochem. 63, 777-822 [CrossRef][Medline] [Order article via Infotrieve]
  25. Larder, B. A., Kemp, S. D., and Darby, G. (1987) EMBO J. 6, 169-175 [Abstract]
  26. L á zaro, J. M., Blanco, L., and Salas, M. (1994) Methods Enzymol. , in press
  27. Méndez, J., Blanco, L., Lázaro, J. M., and Salas, M. (1994) J. Biol. Chem. 269, 30030-30038 [Abstract/Free Full Text]
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  29. Nakamaye, K., and Eckstein, F. (1986) Nucleic Acids Res. 14, 9679-9698 [Abstract]
  30. Peñalva, M. A., and Salas, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5522-5526 [Abstract]
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  32. Rohe, M., Schrage, K., and Meinhardt, F. (1991) Curr. Genet. 20, 527-533 [Medline] [Order article via Infotrieve]
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  34. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130 [Medline] [Order article via Infotrieve]
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  37. Zaballos, A., Lázaro, J. M., Méndez, E., Mellado, R. P., and Salas, M. (1989) Gene (Amst.) 83, 187-195 [CrossRef][Medline] [Order article via Infotrieve]

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