(Received for publication, August 5, 1994; and in revised form, November 28, 1994)
From the
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.
The 29 genome is a linear, double-stranded DNA, with a TP (
)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 TP
dAMP, 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
(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
DXSLYP, KX
NSXYG,
TX
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
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
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
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
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.
To study the truncated
elongation of TPdAMP, the final dATP concentration was varied by
adding different concentrations of unlabeled dATP and keeping constant
the [
-
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
280
0.5 mm), conditions in which the different
TP
(dNMP)
complexes are resolved.
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.
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
. 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).
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).
TPdAMP 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 TP
dAMP 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:
TPdAMP 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 TP
dAMP are
indicated by an arrow.
Figure 6:
Analysis of truncated elongation products
of TPdAMP 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)
is from the right end of
29 DNA, and
TP-d(NMP)
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.
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 (TPdAMP
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
TP
dAMP 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
TP
dAMP 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,
TP
dAMP, 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)
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
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
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, (
)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.