Biochemical Analysis of Point Mutations in the 5'-3' Exonuclease
of DNA Polymerase I of Streptococcus
pneumoniae
FUNCTIONAL AND STRUCTURAL IMPLICATIONS*,
Mónica
Amblar
,
Mario García
de Lacoba,
Maria A.
Corrales, and
Paloma
López
Centro de Investigaciones Biológicas, Consejo Superior de
Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain
Received for publication, September 22, 2000, and in revised form, February 19, 2001
 |
ABSTRACT |
To define the active site of the 5'-3'
exonucleolytic domain of the Streptococcus pneumoniae DNA
polymerase I (Spn pol I), we have constructed His-tagged Spn pol
I fusion protein and introduced mutations at residues
Asp10, Glu88, and Glu114, which are
conserved among all prokaryotic and eukaryotic 5' nucleases. The
mutations, but not the fusion to the C-terminal end of the wild-type,
reduced the exonuclease activity. The residual exonuclease activity of
the mutant proteins has been kinetically studied, together with
potential alterations in metal binding at the active site. Comparison
of the catalytic rate and dissociation constant of the D10G, E114G, and
E88K mutants and the control fusion protein support: (i) a critical
function of Asp10 in the catalytic event, (ii) a role of
Glu114 in the exonucleolytic reaction, being secondarily
involved in both catalysis and DNA binding, and (iii) a nonessential
function of Glu88 for the exonuclease activity of Spn pol
I. Moreover, the pattern of metal activation of the mutant proteins
indicates that none of the three residues is a metal-ligand at the
active site. These findings and those previously obtained with D190A
mutant of Spn pol I are discussed in relation to structural and
mutational data for related 5' nucleases.
 |
INTRODUCTION |
The polymerase function of type-I-like DNA polymerases has been
studied in considerable detail, with biochemical and mutagenesis analysis proceeding in parallel with the structural studies (1-6). Such detailed study have provided a prototypical molecular model of
DNA-dependent DNA polymerization and important insights
into the architecture of the primer and nucleotide binding sites. By contrast, despite the mutational and structural analysis of several 5'-3' exonucleolytic domains of eubacterial polymerases (7-9) and
related bacteriophage 5' nucleases (10-12), the molecular mechanism of
the exonucleolytic reaction still remains obscure.
Sequence comparisons and enzymatic studies indicate that the
eubacterial pol1 I-associated
5' nucleases share significant sequence homology with the
polymerase-independent 5' nucleases from several bacteriophages (13).
The prokaryotic 5' nucleases are also related to mammalian FEN-1
proteins and several yeast proteins of the RAD2 family, having two
large blocks of sequence similarity that bear some resemblance to the
bacterial and bacteriophage nuclease sequences (14, 15). Some clues to
identify important residues in the bacterial 5'-3' exonuclease family
derive from the multiple sequence alignment of 10 bacterial and
bacteriophage nucleases (13). Six conserved sequence motifs containing
14 invariant amino acids were identified, 9 of which were carboxylate
residues. The presence of highly conserved carboxylate residues led to
the proposal that some of these amino acids could be involved in metal
binding at the active site of the 5'-3' exonucleases, as occurs in
other enzymes catalyzing phosphoryl transfer reactions (1). This hypothesis has been further supported by structural data from the 5'
nucleases from bacteriophage T5 (10) and Taq pol (9), and
from T4 RNase H (11). In these three proteins, the 5' nuclease active
site consists of a set of carboxylate residues, which coordinate metal
ligands that are essential for the nuclease activity. However, several
intriguing differences among the three active sites exist, which pose
some important questions.
DNA polymerase I of Streptococcus pneumoniae (Spn pol I) is
a bifunctional protein having two enzymatic activities: DNA polymerase and 5'-3' exonuclease (16). These activities are located on different
domains of the protein that are arranged in the same order in all pol
I-like DNA polymerases (17, 18). Like other DNA polymerases of the
family, both enzymatic activities of Spn pol I are involved in
DNA-repair processes (19, 20). Unlike that of Escherichia
coli (21), the exonucleolytic domain has proved to be essential
for pneumococcal cell viability (20). Previous studies on the
exonuclease activity of Spn pol I showed an essential role for
Asp10 and Asp190 in the exonucleolytic
reaction, since the substitution of these carboxylate residues by Ala
led to an almost total inactivation of the nuclease domain in Spn
polID10A protein (22) and to a drastic reduction of the catalytic
efficiency as well as to an altered metal binding in the Spn polID190A
mutant protein (23).
In this paper, we describe the overproduction and purification of a
His-tagged fusion form of Spn pol I and the introduction of mutations
at three residues of the exonucleolytic domain of the protein:
Asp10 and Glu114 (proposed metal ligands in
other nucleases) and Glu88 (highly conserved among
prokaryotic 5' nucleases). We also report the use of these enzymes in a
kinetic study to explore the roles of the conserved carboxylate
residues in the exonucleolytic reaction. Finally, we present a
three-dimensional model of the putative 5'-3' exonucleolytic domain of
Spn pol I, built by homology modeling, in order to provide us with a
framework to support some structural explanations of the Spn pol I
mutant activities.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes, T4 DNA ligase, and T4
polynucleotide kinase were purchased from New England Biolabs. T7 DNA
polymerase and inorganic pyrophosphatase were obtained from Amersham
Pharmacia Biotech, Taq pol from Roche Molecular Biochemicals, and Pfu
pol from Stratagene. Unlabeled oligonucleotide primers were synthesized in a Gene Assembler (Amersham Pharmacia Biotech) at the Centro de
Investigaciones Biológicas (Madrid, Spain). The E. coli strains used were JM109 (endA1 recA1
gyrA96 thi hsdR17
(rK-
mK+) relA1
supE44 I
(lac-proAB)
F' (traD36
proA+B+
lacIqZ
M15) (24) for cloning experiments and
BL21(DE3) (F- rB-
mB- gal ompT
(int::PlacUV5
T7
gen1 imm21 nin5) (25) for expression
of enzymes to be purified.
Construction of Plasmids Expressing Spn pol I
Derivatives--
The pMA6 plasmid was constructed by cloning a
5.3-kilobase pair EcoRI fragment from plasmid pSM29 (26),
containing the pneumococcal polA gene, into the
EcoRI site of the phagemid pAlter-1 vector (Promega). By
site-directed mutagenesis, two new restriction sites were introduced at
both sides of the polA gene on pMA6 plasmid: EcoRI (between the
10 and
35 boxes) and XhoI
(at the stop codon of the gene). Then, the mutated polA gene
was excised from the pMA6 derivative by digestion at the newly created
EcoRI and XhoI restriction sites and ligated to
the pET21b (fusion vector derived from the E. coli pET5
expression vector; Ref. 27). The resulting plasmid, named pMA9,
contains the polA gene fused at its 3' end to an His tag
coding sequence under the control of T7 gene
10 promoter (27). As a
consequence, the stop codon of the structural gene was replaced by a
DNA fragment encoding the amino acid sequence LE(H)6
obtaining the fusion derivative named Spn pol I-(His), which can be
selectively bounded to chelating Sepharose resin through the histidine
residues. Plasmid pMA10 was derived from pMA9 by removal of the 80-bp
XbaI-EcoRI fragment containing the ribosome
binding site of
10 gene and the T7 tag coding region. Expression
plasmids for pol I derivatives having mutations at the 5'-3'
exonucleolytic domain of the protein, were obtained by swapping the
399-bp EcoRI-NheI restriction fragment from pMA10 for the corresponding PCR mutagenic products. The sequence of the
mutated polA genes was determined by thermal cycle
sequencing of the resulting recombinant plasmids using Taq pol, the
Abitrin 377 automatic sequencer and the corresponding kit supplied by Applied Biosystems Inc., at the Centro de Investigaciones
Biológicas.
Mutagenesis of the polA Gene--
Introduction of the
restriction sites EcoRI and XhoI at the
pneumococcal polA gene was carried out using ECR
(5'-CAATGGTATTTTTTGAATTCTTTCCTTTATA-3') and XHO
(5'-CTGGTACGAGGCTAAACTCGAGGGGGGCTAGTCCTC-3') oligonucleotides, and the
Altered Site in vitro mutagenesis system from Promega.
Mutations of the 5'-3' exonucleolytic domain coding region of
polA gene were obtained by using the high yield method for
site-directed mutagenesis by PCR and three primers as described by
Steingberg et al. (28). The mutagenic primer used was
5'-AAATTATTATTGATTNNNGGGTCTTCTGTAGCT-3', where N corresponds to
degenerated position within the 10th codon, at which the four
nucleotides can be introduced. ECR and A-PCR (5'-AGGGATATTATCCGACTTATCACCCA-3') oligonucleotides were used as
primers for the amplification reaction. PCR products were digested with
EcoRI and NheI and subcloned into the expression
plasmid pMA10. The resulting reaction mixture was introduced in
E. coli JM109 strain by electroporation, and a set of the
clones obtained were sequenced to determine the mutation efficiency.
The PCR mutagenic procedure was first carried out using Taq pol during
the amplification reaction. The analysis of 36 clones showed that 21 of
them contained mutations at non-targeted positions of the
polA gene, such as insertions or deletions (resulting in
frameshift mutations), or multiple base substitutions. Only 4 of the 36 clones contained different mutations (GCG, CAG, CGU, and UAG) at the
10th codon (GAU), giving the amino acid substitutions D10A, D10Q, D10R,
and D10stop, respectively. One of the non-targeted mutations obtained during PCR reactions caused a change at codon 88 (GAG by AAG), resulting in the E88K amino acid substitution in Spn pol I. Then, a
second round of PCR mutagenesis was carried out using Pfu pol (enzyme
with high fidelity of polymerization). Of the 37 clones analyzed, 17 were found to carry plasmids with mutations at the 10th codon.
Moreover, this analysis revealed five new mutations, in which codon
number 10 was replaced by AGU, GUG, ACU, AAA, or GGC. Such changes
correspond, respectively, to the amino acid substitutions D10S, D10V,
D10T, D10K, and D10G. Only one clone contained, in addition to D10A
mutation, the amino acid substitution E114G (change of GAG by GGG at
codon 114). Since Glu114 is highly conserved in the family
of prokaryotic 5'-3' exonucleases (13), the mutation at this position
was subcloned as a single amino acid substitution of Spn pol I into the
expression plasmid pMA10, making use of NdeI and
XhoI sites of polA gene that are bracketing the
114th codon.
Induction Protocol of Spn pol I and Its Derivatives--
Plasmid
pMA10 and its derivatives were transferred to the E. coli
BL21(DE3) overproducer strain. The expression of the wild-type pol I
enzyme and its fusion derivatives was achieved by IPTG induction of the
host strain BL21(DE3) (27) containing the corresponding expression
plasmid (pSM23 for wild-type enzyme (26) and pMA10 set plasmids for
fusion proteins).
Cells containing the corresponding plasmid were grown in M9 medium,
supplemented with 200 µg/ml ampicillin at 37 °C to an absorbance
of 0.45 at 600 nm, and then induced by addition of 0.75 mM
IPTG. Samples were withdrawn at different induction times and crude
extracts prepared to test enzymatic activities. The crude extracts were
also examined by 0.1% SDS, 8% PAGE. Quantification of the proportion
of Spn pol I and mutant forms in crude extracts was performed by
scanning the gels with the Molecular Analyst system (Bio-Rad).
Preparation of Small Scale Crude Extracts--
Cell pastes
obtained from 1.5-ml IPTG-induced cultures of the appropriate strain
were washed by suspension in 1 ml of buffer I (10 mM
Tris-HCl, pH 7.6, 3 mM
-mercaptoethanol), centrifuged, and suspended in 58 µl of lysis buffer II (323 µg/ml lysozyme in
buffer I). The resulting suspensions were incubated 5 min at 37 °C,
supplemented with 92 µl of buffer I, and incubated for another 5 min
at 37 °C. Then, samples were treated with 0.1% Triton X-100 for 5 min at 37 °C and subjected to three cycles of freezing and thawing
at
70 °C and 37 °C, respectively. The viscosity of the extracts
was reduced by passage through a 0.36-mm inner diameter needle;
extracts were then centrifuged and the supernatant stored at
70 °C.
Purification of His Tag Fusion Proteins--
Cells from 100-ml
IPTG-induced cultures were harvested by centrifugation, washed with 50 ml of buffer A (0.5 M NaCl, 20 mM HPO4Na2, pH 7.6), and suspended in 10 ml of the
same buffer. Cell lysis was achieved by incubation with 0.3 mg/ml
lysozyme for 30 min at 0 °C and three cycles of freezing and
thawing. The crude extracts were ultracentrifuged at 81,000 × g, 50 min, and the soluble fraction recovered. After
addition of 10 mM imidazole, the clarified extracts were
added to 4 ml of Chelating Sepharose Fast Flow (Amersham Pharmacia
Biotech), previously charged and equilibrated in buffer A plus 10 mM imidazole. The mixture was incubated by end-over-end
rotation with gentle agitation for 30 min at 4 °C. The resin was
sedimented by centrifugation at 2000 × g, 3 min, and
washed three times by addition of five volumes of 10 mM
imidazole/buffer A and incubation for 5 min at 4 °C as described
above. After washing, the resin was again incubated with two volumes of
40 mM imidazole/buffer A, and the protein was eluted by
incubation with an equal volume of 100 mM imidazole/buffer A. The imidazole was removed from the sample by dialysis against buffer
A plus 50% glycerol, allowing at the same time the concentration and
equilibration in the storage buffer. Quantification of the proteins was
carried out by gel fractionation of the sample and scanning of the gels
with a Molecular Analyst system (Bio-Rad).
Polymerase Assay--
Polymerase activity was determined
on activated calf thymus DNA following the method described previously
(18). One unit of polymerase activity is defined as the amount of
enzyme catalyzing the incorporation of 10 nmol of dNTP into DNA in 30 min at 37 °C.
5'-3' Exonuclease Assays--
The nuclease activity in
DNA-containing 0.1% SDS, 10% polyacrylamide gels after
electrophoresis and removal of SDS was assayed as previously described
by Rosenthal and Lacks (29). Exonuclease activity, assayed using salmon
sperm DNA, was determined in the presence of 0.1 mM
MnCl2 after 30 min at 37 °C, as described previously (16). The salmon sperm substrate, (previously nicked with pancreatic deoxyribonuclease I) was labeled with [3H]dTTP using Spn
polIc269 (an Spn pol I derivative that only contains the polymerase
domain; Ref. 18). Exonuclease activity was also tested using a
5'-32P-labeled (5'-CCAGTCACGACGTTGT-3') or
3'-32P-labeled (5'-CCAGTCACGACGTTGTA-3') oligonucleotide
annealed to M13mp2 ssDNA as described (23). The concentration of DNA
substrate, MnCl2 or MgCl2 and enzymes as well
as reaction time are indicated under "Results." In the case of
reactions with 5' end-labeled substrate, the concentration of mutant
proteins used was as high as possible without exceeding the inhibitory
concentration of 8% glycerol in the reaction (enzymes were stored in
50% glycerol). One unit of exonuclease activity is defined as the
amount of enzyme required for the release of 10 nmol of nucleotide from
DNA in 30 min at 37 °C.
To measure the 5'-3' exonuclease rates, experiments were performed
using the 3'-32P-labeled 17-mer oligonucleotide annealed to
M13mp2 ssDNA as substrate, as described previously (23). The assays
were carried out with the preferred divalent metal ion at the optimal
concentration for the nuclease activity (0.1 mM
MnCl2) and increasing substrate concentrations. The amount
of each enzyme was adjusted to obtain linear conditions, and samples (2 µl) were removed at appropriate times during incubation at 37 °C.
The products were fractionated in a 20% polyacrylamide gel, where the
degradation products are distinguished as discrete bands. A typical
experiment is depicted in Fig. 1. The experimental objective was to
determine the velocity of exonucleolysis
(
n,n
1) by measuring the
proportion of reaction products of n and n
1
nucleotides in the gel. Since every primer that reached position
n
1 also reached position n, the velocity at
the site can be expressed by the following equation.
|
(Eq. 1)
|
t is the reaction time, Ii is
the integrated intensity at site i expressed as a percentage
of total substrate, and m is the length of the final product
of the reaction. The velocity of the conversion reaction
15,14 and
14,13 was measured in order to minimize the contribution of both initial DNA-protein complex formation
and the high dissociation rate at 11-mer product observed for all
proteins analyzed (Fig. 1 and results not
shown). The velocities remained essentially constant for t
up to 30 min for all fusion proteins, indicating that the DNA-enzyme
complexes at the point of analysis were in steady state (data not
shown). In addition, the concentration of the 15-mer and 14-mer
substrates remained lower than 5% of the total DNA, indicating that
only one turnover was measured. The relationship of velocity and
concentration of primed M13mp2 DNA substrate conformed to the
Michaelis-Menten equation, as indicated by linearity in the
Lineweaver-Burk plots (30) of [1/v] versus
[1/primed M13mp2] (Fig. S1). The double-reciprocal plots depicted in
Fig. S1 (available as supplemental material in the on-line version of
this article) were fitted by a linear least-squares regression analysis
and used to determine, from the intercepts, Vmax
(corresponding to the maximum value of
In
1/In) and
Km (corresponding to the value of [primed M13mp2] when In
1/In is at
half-maximum). The catalytic rate of the exonuclease reaction
(kcat) was calculated as
Vmax/[Etotal], ([Etotal] being the concentration of enzyme
used in the assay).

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Fig. 1.
Exonucleolytic reaction catalyzed by Spn pol
I-(His) on 3' end-labeled oligonucleotide annealed to ssDNA from
M13mp2. An autoradiogram is shown of the gel fractionation of the
reaction products obtained from the exonucleolytic reaction performed
with 0.042 unit of exonuclease activity from Spn pol I-(His) and 100 nM DNA substrate. The experiment was carried out as
described under "Experimental Procedures" withdrawing samples at
the times indicated.
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Filter-binding Assays--
Formation of DNA-Spn pol I derivative
complexes was measured by using alkali-treated nitrocellulose filters
(Millipore, type HAWP 45 µm) as described by McEntee et
al. (31). The DNA substrate was obtained by PCR amplification of
the BglII-XbaI 2239-bp polA gene
fragment using ECR and A-PCR oligonucleotides and
[
-32P]dCTP and Taq pol enzyme, which generate dsDNA
with 3' protruding ends. The resulting 616-bp dsDNA was treated with
NheI restriction enzyme, and the 414-bp product, which
contains only one 5' protruding end, was purified by gel
electrophoresis. As the presence of metal ion in the binding buffer was
required to detect specific DNA retention on the filters (data not
shown), it was necessary to standardize conditions of the binding assay
to minimize 5'-3' exonuclease activity. The standard binding reaction
was carried out in 30 µl of buffer B (10 mM Tris-HCl, pH
7.6, 1 mM dithiothreitol, 50 mM KCl, 2.6%
glycerol, 0.1 mM MnCl2) with 0.6 nM
of the
-32P-labeled 414-bp PCR product and increasing
amounts of protein. The mixtures were incubated for 10 min at 15 °C
in order to reach DNA-protein equilibrium and the reactions stopped by
addition of 150 µl of ice-cold buffer B. Samples were filtered,
washed with 9 ml of the same buffer, and dried, and their radioactivity measured by scintillation counting. The quantification of the amount of
Spn pol I derivative-DNA complexes retained on the filters was
corrected by subtracting the nonspecific retention of labeled DNA in
the absence of pol I derivatives and in the presence of an equal bovine
serum albumin concentration.
Molecular Modeling--
A three-dimensional model of the
putative 5'-3' exonucleolytic domain of Spn pol I was built from its
amino acid sequence, the 2.4-Å resolution x-ray structure of the 5'-3'
exonucleolytic domain of Taq pol (9), and 2.5-Å resolution x-ray
structure of the T5 5' nuclease (10) by using knowledge-based protein modeling methods. Their cartesian coordinates were from the Brookhaven Protein Data Bank, with identification codes 1TAQ and 1EXN, respectively. The structural conserved regions and the definition of
the corresponding connecting loops were identified from the multiple
alignment of the amino acid sequences of Spn pol I, Taq pol, and T5 5'
nuclease (Fig. 6). This alignment was initially identified from the
multiple alignment of the nuclease domains of 38 prokaryotic and
eukaryotic proteins (Fig. S2, available as supplemental material in the
on-line version of this article). The overall conformation of the 5'-3'
exonucleolytic domain of Spn pol I was subjected to energy minimization
until convergence, using a combination of steepest descent and
conjugate gradients algorithms. The energy calculations were carried
out under the AMBER force field (33). Computations were performed on a
Power Challenge R10000 by using the BIOSYM software package, release 95.0 (Molecular Simulations, Inc., San Diego, CA).
 |
RESULTS |
Production, Expression, and Purification of Spn pol I-(His) and
Its Derivatives
Spn pol I-(His)--
After a 3.5-h IPTG induction of E. coli BL21(DE3) containing pMA10, Spn pol I-(His) was the major
protein product in cell extract, corresponding to about 9% of the
total protein content (Fig.
2A, lane
4). This yield was very similar to the 10% obtained with
cells harboring pSM23 (26), which encodes Spn pol I (Fig. 2A, lane 3). The nuclease activity in
cell extracts from both BL21(DE3)[pMA10] and BL21(DE3)[pSM23]
cultures, was detected in situ using a DNA containing
polyacrylamide gel (Fig. 2B). In both extracts, a
degradation band was detected corresponding to a polypeptide of ~100
kDa, the predicted size for Spn pol I and its fusion derivative (Fig.
2B, lanes 2 and 3). In
addition, other bands of activity were observed that presumably
corresponded to proteolytic fragments of the pneumococcal enzymes,
since they were not detected in extracts carrying the pET5 vector (Fig.
2B, lane 1). Quantification of the
amount of Spn pol I and Spn pol I-(His) (Fig. 2A,
lanes 3 and 4) versus their
nuclease activity (Fig. 2B, lanes 2 and 3) revealed that the fusion of the His tag did not
affect the 5'-3' exonuclease specific activity. However, this fusion
resulted in a 5-fold reduction of the polymerase activity of the
pneumococcal enzyme (producing 60 units of polymerase activity/mg of
protein after a 3.5-h induction, compared with 318 units for the vector coding for the wild-type Spn pol I, after correction for the endogenous polymerase activity (3 units) of the host organism). Thus, the Spn pol
I-(His) construct is appropriate for mutational analysis of the 5'-3'
exonuclease activity of the pneumococcal enzyme.

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Fig. 2.
Overexpression and purification of Spn pol
I-(His). 15 µg of crude extracts from 3.5-h IPTG-induced
cultures of E. coli BL21(DE3) carrying the indicated
plasmids were fractionated in a 8% polyacrylamide gel. The protein
content was analyzed by Coomassie Brilliant Blue stain (A),
and the nuclease activity of the extract was assayed by DNase gel assay
(B). The purity of Spn pol I-(His) at each purification step
was evaluated by 0.1% SDS, 8% PAGE and staining the gel with
Coomassie Brilliant Blue (C). Lane C,
225 ng of purified Spn pol I; lane 1, 1/1400
volume of crude extract; lane 2, 1/1400 volume of
the sample applied to chelating Sepharose resin; lane
3, 1/1400 volume of the sample recovered after application
to the resin; lanes 4-6, 1/600 volume of each of
the three wash steps with 10 mM imidazole; lane
7, 1/500 volume of the wash step with 40 mM
imidazole; lane 8, 1/250 volume of the eluted
fraction with 100 mM imidazole. St, mixture of
polypeptides of known molecular weight.
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Spn pol I-(His) was purified from IPTG-induced cultures by binding to a
chelating Sepharose resin (Fig. 2C). By testing different concentrations of imidazole-eluting agent, we developed a purification procedure yielding 0.37 mg of Spn pol I-(His)/liter of induced culture.
The purity of the protein sample was greater than 60% (Fig.
2C, lane 8), with polymerase and
exonuclease specific activities of 614 and 1049 units/mg of protein, respectively.
5'-3' Exonuclease Mutant Forms of Spn pol I-(His)--
The
Asp10 of Spn pol I is highly conserved among the 5'
nucleases (7), and structural data support the involvement of this residue in the active site (9-11). Substitution of Asp10
by Ala in Spn pol I (22) or its equivalent residues in Eco pol I (7) or
Mtb pol I (8) by Asn, yielded proteins with so little 5' nuclease
activity that they could not be further analyzed. To obtain a mutant
protein that could be kinetically characterized, we mutated the
pneumococcal polA gene at the 10th codon. Ten different
mutant forms of Spn pol I-(His) were obtained, eight of them with
different amino acid changes at the Asp10 (D10G, D10V,
D10T, D10K, D10A, D10Q, D10S, and D10R) and two of them containing
either E88K or E114G amino acid substitutions. These carboxylate
residues, Glu88 and Glu114, are also conserved
among the prokaryotic 5' nucleases (7). All mutant forms were
overproduced in E. coli BL21(DE3) and purified as for Spn
pol I-(His). Analysis of the protein production and polymerase activity
after IPTG induction at 30 or 37 °C of two representative mutant
derivatives (Spn polID10A-(His) and Spn polID10G-(His)) revealed that
incubation for 3 h at 37 °C was optimal induction, without
significant insolubilization of the proteins (data not shown). This was
surprising, since we have observed previously that 91% of Spn polID10A
was insoluble upon induction at 37 °C (22). This procedure allowed
us to purify the mutant proteins with a typical yield of 0.4 mg of
protein/liter of induced culture at greater than 70% purity and
200-600 units of polymerase activity/mg of protein.
Enzymatic Activities of Spn pol I-(His) and Its
Derivatives--
All the purified proteins were assayed for polymerase
activity on activated DNA (Table I).
Similar specific activities were obtained for all mutant and control
fusion proteins, indicating that the polymerase domain of Spn pol I was
functionally unaffected by the single amino acid changes in the 5'-3'
exonucleolytic domain. Next, we determined the exonuclease activity of
the enzymes on salmon sperm DNA substrate. Only E88K and the control
protein showed detectable activity: 890 and 1000 units/mg of protein, respectively. Due to the low exonuclease activity of the mutant proteins, it was necessary to use a more sensitive assay to determine the effect of the mutations introduced at the exonucleolytic domain. Therefore, a 5'-32P-labeled oligonucleotide annealed to
M13mp2 ssDNA was used as substrate to compare the 5'-3' exonuclease
activity of Spn pol I-(His) and all the mutant proteins (Fig.
3). With this substrate we observed the
first nucleolytic event in the exonucleolytic reaction. Spn pol I-(His)
generated the same products as those previously obtained with Spn pol I
(23), derived from both the 5'-3' exonuclease (mononucleotides) and the
5' end-dependent endonuclease activities (dinucleotides),
which are present in all 5' nucleases of the family (34). Of the 10 mutant proteins, only those carrying the mutations E114G, E88K, D10G,
and D10A gave detectable activities, with that of the Spn
polID10A-(His) mutant only being visible after 16 h of incubation,
as observed previously with the Spn polID10A mutant (22). In addition,
none of these four mutations altered the ratio of mononucleotide to
dinucleotide products (~15:1), in contrast to the previously
described behavior of Spn polID190A, which showed a prevalence of
endonucleolytic over exonucleolytic cleavages (23). In some reactions,
products most likely generated by removal of mononucleotides at the 3'
end of the oligonucleotide substrate were also observed. These products
were presumably produced by residual contamination of the samples with
3'-5' exonucleases. Quantification of the experiments, shown in Fig. 3,
allowed us to categorize the mutant proteins in terms of their
exonuclease activity (Table I). The E88K mutant possessed 26% of the
exonuclease specific activity present in Spn pol I-(His), indicating
that Glu88 is not an essential residue for this enzymatic
activity. By contrast, the mutations introduced at Glu114
or Asp10 resulted in a decrease of more than 98% of the
exonuclease activity compared with that of the control enzyme. In the
case of the amino acid substitutions of Asp10, 5'-3'
exonuclease activity was only measurable in D10G and D10A mutants.
These results argue in favor of an essential role of the
Asp10 and Glu114 residues in the exonuclease
activity. Therefore, we proceeded to determine the role played by
Asp10, Glu88, and Glu114 residues
by analyzing the mutant forms Spn polID10G-(His) (the only mutant at
Asp10 that retains significant exonuclease activity), Spn
polIE88K-(His), and Spn polIE114G-(His).
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Table I
Specific enzymatic activities of His tag fusion proteins
Polymerase activity was assayed on activated calf thymus DNA. The 5'-3'
exonuclease activity was determined from the experiments performed with
5'-32P-end-labeled oligonucleotide annealed to M13mp2 ssDNA.
The enzymatic activities are expressed as units/µg of protein
(units · µg 1). The figures are the average of at
least three independent experiments. Standard deviations are indicated.
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Fig. 3.
Exonuclease activity of His tag fusion
proteins on 5' end-labeled oligonucleotide annealed to M13mp2
ssDNA. Reaction mixtures contained 1.5 pmol of DNA and 1 mM MnCl2. Samples were removed at 10 min, 30 min, 1 h, 2 h, and 16 h. The concentration of the
enzymes in the reaction were: 0.05 pmol of Spn pol I-(His) 2.2 pmol of
Spn polIE114G-(His), 0.18 pmol of Spn polIE88K-(His), 0.26 pmol of Spn
polID10S-(His), 0.62 pmol of Spn polID10G-(His), 0.096 pmol of Spn
polID10V-(His), 0.12 pmol of Spn polID10T-(His), 0.33 pmol of Spn
polID10K-(His), 0.91 pmol of Spn polID10A-(His), 0.35 pmol of Spn
polID10Q-(His), and 0.21 pmol of Spn polID10R-(His). C,
control; WT, Spn polI-(His); E114, polIE114G;
E88, polIE88K; D10, polID10V.
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Metal Dependence of the 5'-3' Exonuclease Activity of His Tag Spn
pol I Derivatives--
The divalent metal ion requirements for the
nuclease activity of the His tag Spn pol I fusion proteins were
determined using a 3'-32P-end-labeled M13-primed substrate
and measuring the 5'-3' exonuclease activity at different
Mn2+ or Mg2+ concentrations. The reaction
products were fractionated in a denaturing 20% polyacrylamide gel and
the deg-radation bands quantified as
described previously (23).
The results obtained revealed that the Mn2+ and
Mg2+ dependence of the exonuclease activity was similar for
the four fusion proteins analyzed (Fig.
4), and correlated with that previously
obtained for Spn pol I (23). Thus, neither the fusion of the His tag at
the C-terminal end of the protein nor the amino acid changes introduced
altered the metal dependence of the 5'-3' exonuclease activity of the
S. pneumoniae enzyme. The His tag proteins showed maximum
exonuclease activity over a wide range of Mn2+
concentration, ranging between 10 µM and 1 mM
(or even 5 mM) MnCl2, but diminished
dramatically at higher concentrations. In the case of Mg2+,
the exonuclease activity of the four fusion proteins increased gradually from the lowest concentration tested (5 µM
MgCl2), reaching its maximum value at 10 mM,
above which the activity drastically decreased. The diminution of
exonuclease activity observed at high metal ion concentration for all
proteins was probably due to the metal binding to the substrate, rather
than to an inhibitory effect on the protein.

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Fig. 4.
Metal dependence of the exonuclease activity
of fusion proteins. Reactions were performed with 3 pmol of
3'-32P-end-labeled oligonucleotide annealed to M13mp2 ssDNA
and increasing concentrations of MgCl2 or
MnCl2. To determine the exonuclease activity in the
presence of Mg2+, the incubation time was 30 min for
reactions catalyzed by Spn pol I-(His) and Spn polIE88K-(His), or
2 h for those catalyzed by the Spn polID10G-(His) and Spn
polIE114G-(His). Reaction time in the presence of Mn2+ was
10 min for Spn pol I-(His) and Spn polIE88K-(His), 30 min for Spn
polIE114G-(His), and 2 h for Spn polID10G-(His).
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Comparative Kinetic Analysis of His Tag-Spn pol I Fusion
Proteins--
To investigate the role of the Asp10,
Glu88, and Glu114 residues in the 5'-3'
exonuclease activity of Spn pol I, we determined the kinetic parameters
of the D10G, E88K, and E114G mutant enzymes and of Spn pol I-(His)
control protein (Table II). The apparent catalytic rate (kcat) and Km
values were calculated from experiments performed using
3'-32P-end-labeled 17-mer oligonucleotide annealed to
M13mp2 ssDNA as substrate for the exonucleolytic reaction. The scheme
of the reaction is as follows.
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Table II
5'-3' exonuclease kinetic constants of His tag fusion proteins
Exonuclease rates were measured at different substrate concentrations
as indicated under "Experimental Procedures." The
Km and kcat values are the
average of those obtained for the two catalytic events analyzed in Fig.
S1. The KD values were determined from filter
binding assays as described in the text. ND, not determined.
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E represents the enzyme, Dn is
the original 17-mer primer bound to DNA, Dn
1,
Dn
2, Dn
3, and Dn
4 are the products of
excision of one by one mononucleotide (dNMP), and
Dm is the final product of the reaction. The side pathways
(downward pointing arrows) lead to dissociation of complexes
E·Dn
1,
E·Dn
2, E·Dn
4, with rate constants
k
a, k
b,
k
d. The reverse association of the
enzyme and the reaction products (Dn
1, Dn
2, and Dn
4) can
occur via ka, kb,
kd (upward pointing arrows).
In order to minimize the contribution of both initial DNA-protein
complex formation
(k
0/k0) and the high
dissociation rate at 11-mer product (kdis) (see
Fig. 1), we measured the velocity of the conversion of 15-mer substrate
to 14-mer product (
15,14) and of 14-mer substrate to
13-mer product (
14,13) (see details under
"Experimental Procedures"). In addition, association of the enzyme
and the substrates of the reactions analyzed
(D15 and D14) was
minimized, since at the time periods assayed the concentration of
D15 and D14 was very
small in relation to original primer (D17) (see
Fig. 1). Moreover, correction for further conversion of both substrate
and product to shorter products (see details under "Experimental Procedures") allow us to neglect association and dissociation rates
downstream of the target reaction. The Lineweaver-Burk plots of
[1/v] versus [1/primed M13mp2] are depicted
in Fig. S1 (available on-line as supplemental material to this
article), and they were used to calculate the kinetic constants shown
in Table II. The apparent Km and
kcat values were similar for the two catalytic
events analyzed for each protein except for the E88K mutant, where the
rate of conversion of D14 to
D13 (1.25 s
1) was
slightly higher than that of D15 to
D14 (0.53 s
1). This
indicated that the kinetic constants were not significantly affected by
the nature of the nucleotide present at the 5' end of each substrate
(dAMP or dGMP). The apparent Km of the E88K mutant
was similar to that of Spn pol I-(His), and this mutation resulted in
only a 5-fold reduction of the apparent kcat. These results indicate that Glu88 is not essential for the
exonucleolytic reaction. By contrast, the D10G and E114G mutants
possessed kcat values ~6000-fold and ~100-fold lower than that of Spn pol I-(His), respectively. This sharp reduction of the apparent kcat values
suggested an important role for Asp10 and
Glu114 residues in the exonuclease catalytic event. The
apparent Km value of D10G and E114G were lower than
that of the control protein, implying that these mutations resulted in
a higher DNA binding affinity. However, it is important to keep in mind
that Km is not an equilibrium dissociation constant
(KD) because it is also affected by
kcat. Moreover, in our kinetic analysis of a
catalytic even x, we can assume that the apparent kinetic constant measured is Km = (k
x + kcat)/kx. Therefore, the fast catalytic rates of Spn pol I-(His) and E88K mutant
could mask the actual affinity for the substrate yielding the high
apparent Km values observed. Supporting this hypothesis, the Km previously obtained for Spn pol I (Km = 100 nM; Ref. 23) was also lower
than that for Spn pol I-(His), probably due to the low
kcat of wild-type enzyme (kcat = 0.11 s
1; Ref.
23). The differences in kcat between both
wild-type and Spn pol I-(His) enzymes can be explained because of the
different purification procedures used. The His tag fusion proteins
were obtained using a 1-day purification step, whereas Spn pol I had been previously purified over 1 week using a more complex protocol consisting of three chromatographic steps (23). Therefore, the inactivation of the wild-type enzyme was probably higher than that of
the fusion proteins. This was borne out by the fact that the specific
exonuclease activity of the enzymes prepared for the kinetic
experiments were 14 units/nmol for Spn pol I-(His) and 1 unit/nmol for
Spn pol I. In conclusion, the kinetic analyses allowed the
determination of the catalytic rate of the enzymes, but the apparent
Km values were not a measure of the DNA binding
capability of these His tag fusion proteins. Therefore, we proceeded to
determine the dissociation constant (KD) to
establish whether the substrate affinity was affected by the mutations.
DNA Binding Affinity of His Tag Fusion Proteins--
The effect of
the mutations in Spn pol I-(His) on DNA-exonucleolytic domain
dissociation equilibrium constant (KD) was measured
by filter binding assays using homogeneously 32P-labeled
dsDNA substrate. The retention of DNA in the filters requires the
interaction of the DNA with the protein. Since Spn pol I possesses two
enzymatic domains, for which DNA is a substrate, either domain could
bind DNA. To minimize interference of DNA interactions with the
polymerase domain on binding of the substrate to the exonucleolytic
domain of Spn pol I, we used assay conditions (see "Experimental
Procedures") in which no DNA binding was detected with Spn polIc269
(data not shown). This protein only contains the polymerase domain of
Spn pol I and possesses the same Km for DNA as the
wild-type protein (18).
Complex formation between each protein and DNA was measured as a
function of protein concentration, and the saturation curves are
depicted in Fig. 5. The data were fitted
by direct nonlinear least-squares regression program to the equilibrium
binding Equation 2 for independent sites.
|
(Eq. 2)
|
[C], [D] and [P] represent concentrations of formed complex,
DNA, and free protein, respectively, K is the apparent
dissociation rate constant (KD), and n is
the Hill coefficient. Transformation of Equation 2 into a linear form
yielded Equation 3.
|
(Eq. 3)
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The Hill plots of the data log([C]/([D]-[C]))
versus log[P] are depicted in Fig. S3 (see supplemental
material, available on-line). The Hill coefficient n for the
protein tested ranged from 0.8 to 1.2, indicating a stoichiometry of
1:1 for the protein-DNA complexes (as expected since the DNA substrate
contains only one 5' protruding end). Therefore, the apparent
KD of Spn pol I-(His) as well as of D10G, E88K,
E114G, and D10K (which displayed a non-measurable 5'-3' exonuclease
activity) mutant enzymes was calculated from Equation 4 at each protein
concentration tested, and the averages are shown in Table II.
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(Eq. 4)
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Spn pol I-(His) and the D10G, D10K and E88K mutant enzymes showed
a similar KD value, ranging between 7 and 14 nM. These results show that differences of more than
6000-fold on the catalytic rate of the proteins are not reflected in
the binding assay. Moreover, the results indicate that neither
Asp10 nor Glu88 are involved in DNA binding.
However, the E114G mutation produced a 13-fold increase of the
KD, suggesting a direct involvement of
Glu114 in DNA binding of Spn pol I through its
exonucleolytic domain.

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Fig. 5.
Detection of DNA-protein complex formation by
filter assay. The binding reactions with the indicated proteins
were performed as described under "Experimental Procedures."
Concentrations of retained DNA-protein complexes [C], [P] obtained
upon increasing protein concentration are depicted. Experimental data
were treated through a non-linear regression analysis program (Curve
Expert 1.3, Microsoft Corp.).
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 |
DISCUSSION |
In recent years many structural and mutational data have
emerged from studies on different 5' nucleases, showing that catalysis is supported by divalent metal ions whose ligands are carboxylate residues that are highly conserved in all 5' nucleases. A sequence alignment from 10 bacterial DNA polymerases and related
bacteriophage 5' nucleases (13), revealed the presence of 10 invariant
or highly conserved carboxylate residues, 9 of which appear to be important for the exonucleolytic reaction (reviewed in Ref. 1). The
subsequent inclusion in the analysis of eight sequences of polymerase-dependent and independent 5' nucleases from
different bacteria, plus the comparison of the 5' nuclease domain of
Eco pol I and the eukaryotic FEN-1 enzyme, revealed that only six invariant residues are present in all prokaryotic and eukaryotic nucleases (7). Moreover, the multiple alignment of the 5' nuclease domain of 38 prokaryotic and eukaryotic proteins (Fig. S2 (see on-line
supplemental material)) allowed us to confirm and further support the
previously observed conservation patterns of acidic residues.
We therefore carried out a mutational analysis of the 5' nuclease
domain of Spn pol I-(His), to gain insights into the structure-function relationships in 5' nucleases. Three carboxylate residues of the 5'
nuclease domain of Spn pol I were analyzed: Asp10,
Glu88, and Glu114. Although Glu88
is not an invariant amino acid, in 82% of the sequences aligned, this
position is occupied by an acidic residue (Fig. S2). In addition, some
of the amino acids surrounding Glu88 are invariant and have
been shown to be important for the 5' nuclease activity of Eco pol I
(35) and 5' nuclease from T5 (36, 37). Therefore, it has been proposed
that the Glu88 region could be involved in DNA binding (7).
However, our results revealed that substitution of Glu88 by
Lys did not drastically alter either the catalytic constants or the DNA
binding ability of Spn pol I. Thus, it seems that Glu88
specifically does not play an essential role in the exonucleolytic reaction.
The crystal structures of Taq pol, T5 nuclease, and T4 RNase H, and the
related eukaryotic flap endonuclease MjFEN-1 have been
reported recently (9-11, 38). Although they differ in detail, all four
share an active site made up of conserved residues that coordinate, at
least, two divalent metal ions. Despite the high identity of the
residues that constitute the active site, differences exist in the
number of metals bound and the distances between them. The 5' nuclease
from T5, T4 RNase H, and MjFEN-1, contain two divalent metal
ions separated by 5-8 Å, whereas in the exonucleolytic domain of Taq
pol, three different metal binding sites were detected: site I
(crystals soaked in Zn2+), and sites II and III (crystals
soaked in Mn2+). Sites I and II are separated by about 5 Å and are each about 10 Å from site III. The 5-Å distance between sites
I and II in Taq pol is similar to the 3.9 Å that separates the two
metal ions bound to the 3'-5' exonucleolytic domain of the Klenow
fragment (39). Accordingly, a two-divalent metal ion mechanism,
analogous to that used by the 3'-5' proofreading exonuclease of Klenow
fragment, was proposed for the 5'-3' exonuclease of Taq pol (7, 9). Such a mechanism implies that two divalent metal ions (I and II) promote the formation of a hydroxyl ion that attacks the scissile phosphodiester bond (MgA) and stabilize the oxyanion leaving group (MgB) and the pentavalent transition state formed during the reaction (MgA and MgB).
The two divalent metal ions bound at the active site of T4 RNase H and
5' nuclease from T5 are further apart (7 and 8 Å, respectively). This
greater distance makes it difficult to propose a mechanism similar to
that of the 3'-5' exonucleases. It is more likely that the divalent
metal ions act independently in the exonucleolytic reaction, one of
them (located at Me1 metal binding site) being essential for catalysis
and the other (at the Me2 metal binding site) playing a more indirect
role, probably in substrate binding (11).
The alignment of the 5'-3' exonuclease domains of Spn pol I and Taq
pol, and the 5' nuclease from bacteriophage T5 (derived from the
multiple alignment of the 38 nucleases depicted in Fig. S2 (see on-line
supplemental materials)) revealed a high sequence homology, Spn pol I
possessing a 62% and a 51% similarity with Taq pol and T5,
respectively (Fig. 6). This homology
allowed us to build a three-dimensional model of the exonucleolytic
domain of Spn pol I (residues 1-290) based on the crystal structures of the Taq pol and the T5 enzyme. The model predicts that
the 5'-3' exonucleolytic domain of Spn pol I should adopt a overall conformation similar to that of those nucleases, consisting of a
central
-sheet, surrounded by clusters of helices on either side,
and organized in two subdomains. The superimposition of the
three-dimensional model on the structure of the 5'-3' exonucleolytic domain of Taq pol (Fig. 7A)
and T5 5' nuclease (Fig. 7B) clearly revealed that most of
the secondary structural elements are conserved in all three proteins
(particularly between Spn pol I and Taq pol) and that they are largely
present in the same relative positions. Another interesting feature of
the Spn pol I model is the presence of a helical arch similar to that
of the T5 nuclease, which has been proposed as being important for the
threading mechanism of the structure-specific endonuclease activity of
these types of enzymes (10). In our model, the helical structure is
composed of two helices made up of 36 residues, smaller than the
three-helix arch of 44 residues present in the bacteriophage nuclease
(the corresponding region in Taq pol is crystallographically
disordered; Ref. 9).

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Fig. 6.
Amino acid sequence alignment of the 5'
nuclease from T5 and the 5'-3' exonucleolytic domains of Spn pol I and
Taq pol. This alignment was derived from the multiple
sequence alignment of 38 exonucleolytic domains of the family A of DNA
polymerases, and prokaryotic and eukaryotic nucleases (see Fig. S2,
available as supplementary material on-line ). The numbers
indicate the amino acid position relative to the N terminus of each
sequence. Invariant residues are shown as white
letters over black background, whereas
similarity is indicated by gray background.
Conserved secondary structural motifs ( -helices as hollow rectangles
and -strands as arrows) are indicated at the
top of the aligned sequences. Invariant acidic residues at
the active site are indicated at the bottom of the aligned
sequences. , residues present in both prokaryotic and eukaryotic
nucleases; , residues exclusively present in the prokaryotic
enzymes; , acidic residue conserved in prokaryotic enzymes.
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Fig. 7.
Structural modeling of the putative 5'-3'
exonucleolytic domain of Spn pol I. Solid
ribbon represents the optimally superimposed
polypeptide backbone of the energy-minimized model of the 5'-3'
exonucleolytic domains of Spn pol I (red) and Taq
pol (green) (A) or 5' nuclease from T5
(cyan) (B). The amino acid side chains bound to
metal ions at the active site of Taq pol (A) or
T5 5' nuclease (B) are drawn with solid
sticks (yellow). Nt and Ct
indicate the N- and C-terminal ends, respectively.
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The predicted active site of the nuclease domain of Spn pol I
(Fig. 8) is located in a similar position
to those of the crystallized enzymes. The nine acidic residues
conserved in the prokaryotic nucleases (Fig. 6; see also Fig. S2 in
on-line supplementary material), including the Asp10 and
Glu114, cluster in a sphere with 11-Å radius that should
accommodate the divalent metal ions. Similar radii are observed in the
spheres containing the analogous residues in 5' nuclease from T5 (9 Å) and Taq pol (10.5 Å), indicating that, in an analogous
manner, two or three metal ions could be bound at the active site of
Spn pol I exonuclease.

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Fig. 8.
Close-up view of the active site of the
structural model of Spn pol I 5'-3' exonucleolytic domain. The
polypeptide backbone of Spn pol I is represented as a solid
ribbon (cyan). The conserved-amino acid side
chains are represented as solid sticks with the
carbon atoms in green and the oxygen atoms in
red. The seven residues present in both prokaryotic and
eukaryotic nucleases are labeled in yellow, and those
exclusively present in the prokaryotic enzymes are labeled in
white.
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From our model of Spn pol I, we can also infer that Asp190
(residue conserved among all prokaryotic 5' nucleases and analyzed previously by the mutation D190A; Ref. 23) is located at the active
site of the exonucleolytic domain. Analysis of this mutant showed that
the replacement of Asp190 by Ala produced a sharp reduction
of the kcat for the exonuclease activity, which
was accompanied by a deficiency in catalytic DNA-protein complex
formation as well as a marked preference of the protein for
Mn2+ over Mg2+ and an alteration of the optimal
concentration of Mn2+ for catalysis (23). These data
suggest that Asp190 is an essential residue, being involved
simultaneously in DNA binding and catalysis, and that its function is
mediated by the metal coordination at the active site. The crystal
structure of T5 nuclease showed that Asp201 (counterpart of
Asp190) together with Asp153,
Asp155, and Asp204 (counterparts of
Asp139, Asp141, and Asp193 in Spn
pol I) are within coordination distance of the metal ion located at the
Me2 site, which has been implicated in substrate binding.
Superimposition of the active site of the Spn pol I model on the
structure of the T5 enzyme (data not shown),revealed a RMS deviation
between the Asp190-Spn pol I residue and the bacteriophage
nuclease Asp201 of only 2.6 Å, supporting the involvement
of Asp190 in the coordination of the metal ion bound at the
putative Me2 site in Spn pol I.
It can be also inferred from our model that Asp10 and
Glu114 could coordinate the metal ions bound at the active
site, so that these residues, which are present in all prokaryotic and
eukaryotic 5' nucleases, could be essential for its nuclease activity.
The available structural data indicate that these residues are involved in binding a divalent metal ion at the active site (Asp10
in Me1 of T5 nuclease and site I of Taq pol, and Glu114 in
Me1 of T5 and site III of Taq pol). However, our results show that
substitution of Asp10 or Glu114 by Ala
drastically reduces exonuclease activity of Spn pol I without changing
its Mn2+ and Mg2+ dependence. Thus, they
indicate that these residues do not coordinate metal ions.
Nevertheless, they do not rule out that, upon removal of one of these
putative ligands, metal ions can remain bound to the active site of the
exonucleolytic domain of Spn pol I through interactions with other
acidic residues, although an alteration of affinity for the metals
should be expected, as was the case for the D190A mutant of Spn pol I
(23). Moreover, in our structural model of Spn pol I,
Asp64, Asp116, and Asp117 (metal
ligands in other 5' nucleases) cluster within a sphere of ~7.5-Å
radius (Fig. 8) and the conformational freedom of their side chains
could allow an arrangement to bind a divalent metal ion at the putative
Me1 site. Therefore, at least, we can infer that Asp10 and
Glu114 do not have a crucial role in metal coordination at
the nuclease active site of Spn pol I.
The essential role of Asp10 in exonuclease activity is
unquestionable, since mutations at this position in all nucleases
studied led to an almost total inactivation of the protein, as reported for D13N mutation in Eco pol I (7), D21N in Mtb pol I (8), D34A in
hFEN-1 (40), and D19N in T4 RNase H (12). We have demonstrated that, in Spn pol I, the Asp10 is involved
exclusively in the catalytic event of the exonucleolytic reaction,
because the substitution D10G produced a ~6000-fold reduction of the
catalytic rate without affecting the DNA binding capability. A similar
role has been proposed for the analogous Asp34 residue in
hFEN-1, since its D34A mutant is inactive in cleavage but
not in DNA binding (40). Our experimental results suggest that the
Asp10 in Spn pol I is not critical for metal coordination.
Therefore, this residue could be essential by acting like a general
base during the reaction, activating a water molecule and generating the hydroxyl group required for nucleophilic attack. For this purpose,
Asp10 must be in close proximity to the metal ion bound to
the putative Me1 site, as shown by our nuclease domain model of the
pneumococal enzyme. This metal should stabilize the pentavalent
transition state formed during the reaction and therefore would be
essential for catalysis.
The role played by Glu114 in exonucleolytic reaction is
less clear. This amino acid of Spn pol I, as well as the equivalent
residue of hFEN-1 (40), seems to be involved in substrate
binding, and has been considered less essential than other residues
analyzed for the exonuclease activity of Eco pol I (7) and Mtb pol I (8). The structural data are highly contradictory, since the counterpart of Glu114 seems to be involved in binding of
the catalytic metal ion (Me1) in T5 nuclease (10) and
MjFEN-1 (38), does not interact with metal ions at the T4
RNase H active site (11), and is part of the metal binding site III in
Taq pol (for which a nonessential role has been proposed (9)). Thus, it
is likely that Glu114 of Spn pol I will be at the
exonuclease active site, as shown by the Spn pol I exonuclease
conformational model, contributing to the extensive
hydrogen-bonding/electrostatic network surrounding both metal ions,
rather than interacting in a direct or indirect manner with any one of
them. If this hypothesis is correct, substitution of Glu114
by Gly should not produce a loss of a coordination with one or two
metal ions (as indicated by our results) but a reorientation of the
metal(s) at the active site, making the catalytic center less
accessible to the DNA substrate and reducing the catalytic efficiency of the reaction.
Summarizing the above results and the available data from other 5'
nucleases, it seems that the involvement of two metal ions playing
different roles is the more plausible mechanism for exonucleolytic reaction in 5' nucleases. A water molecule (probably activated by
Asp10 or an equivalent residue) would be responsible for
the nucleophilic attack of the phosphodiester bond. The divalent metal
ion bound to the Me1 site would be essential for the reaction,
stabilizing the generated transition state, whereas the metal ion bound
to the Me2 site positions the DNA correctly at the active site for cleavage (probably by interacting with the 5' end). Thus, the role
played by the Me2 site would be also important for the reaction, since
an incorrect orientation of the DNA at the active site would lead to a
drastic reduction of the exonuclease activity. Therefore, its function
may not be as indirect as proposed previously (8, 12). This mechanistic
model does not necessary exclude the possibility of a reaction similar
to that of the 3'-5' exonuclease of the Klenow fragment proposed for
Taq pol. The 5' nucleases are able to recognize a wide variety of DNA
substrates with different structures and to catalyze endo- or
exonucleolysis; thus, they must possess a very flexible active site
capable of adopting different conformations depending on the substrate
and the reaction taking place. It is possible that the differences of
active site architecture seen in distinct 5' nucleases reflect the
different functional conformations that these active sites can adopt.
For example, in the case of Taq pol, one metal binding site was
detected in presence of Zn2+ whereas two different sites
appeared in presence of Mn2+. This could be taken as an
indication of the flexibility of its active site, being
capable of adopting in vivo two different conformations depending of the requirements of the enzyme. By contrast, it is also
possible that the differences between distinct nucleases are a
consequence of the individual substrate specificities of the enzymes.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. Espinosa, Dr. L. Blanco, and
Dr. S. W. Elson for helpful discussions and critical reading of
the manuscript. We are grateful to Drs. T. A. Steitz and S. H. Eom for providing the refinement of the tertiary structure of Taq
pol and to Dr. A. Ceska for the initial model of the active site
of Spn pol I, based in the three-dimensional structure of T5 nuclease,
and for helpful discussion.
 |
FOOTNOTES |
*
This work was performed under the auspices of the Consejo
Superior de Investigaciones Científicas and was supported by
European Union Grant QLK2-CT-2000-00543 and by the Program of Strategic Groups of the Comunidad de Madrid.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. S1-S3.
To whom correspondence should be addressed. Instituto de
tecnologie Química e Biológica. Universidade Nova de Lisboa,
Apart. 127, 2781-901 Deiras, Portugal. Tel.: 351-214469548; Fax:
351-214411277.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M008678200
 |
ABBREVIATIONS |
The abbreviations used are:
pol, polymerase;
Spn
pol I, S. pneumoniae DNA polymerase;
Spn polID10A, Spn pol I
with the amino acid substitution D10A;
Spn polID190A, Spn pol I with
the amino acid substitution D190A;
FEN, flap endonuclease;
Taq pol, Thermus aquaticus DNA polymerase I;
Pfu pol, Pyrococcus furiosus DNA polymerase II;
IPTG, thio-
-D-galactoside;
Spn pol I-(His), Spn pol I fused to
an His tag at the C-terminal end;
Eco pol I, E. coli DNA
polymerase I;
Mtb pol I, Mycobacterium tuberculosis DNA
polymerase I;
MjFEN-1, Methanococcus jannaschii
FEN-1;
hFEN-1, human FEN-1;
bp, base pair(s);
PCR, polymerase chain reaction;
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA.
 |
REFERENCES |
1.
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