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J Biol Chem, Vol. 274, Issue 35, 25151-25158, August 27, 1999
From the Department of Biological Science, University of Alberta,
Edmonton, Alberta T6G 2E9, Canada
The 3' Many DNA polymerases have the ability to proofread newly
replicated DNA by transferring the 3'-end of the primer-strand from the
polymerase to the 3' The two-metal ion mechanism may extend to other enzymes that catalyze
phosphoryl transfer, for example, bacterial alkaline phosphatase, RNase
H of the human immunodeficiency virus reverse transcriptase,
single-stranded P1 nuclease, and phospholipase (reviewed in Ref. 15).
As observed for E. coli DNA pol I, a distance of about 3.9 Å separates two essential metal ions in the active centers of these
enzymes. These observations led Steitz and Steitz (15) to propose that
RNA molecules involved in hydrolysis and splicing reactions may
similarly position two divalent metal ions to carry out phosphoryl
transfer reactions. A two-metal ion model for the hammerhead ribozyme
mechanism is illustrated in Fig. 2, but catalysis does not require a
metal hydroxide ion (16, 17), although single-metal-hydroxide-ion
models have been proposed (reviewed in Ref. 18). Both one- and
two-metal ion models for hammerhead ribozyme activity require the
ribose 2'-OH group, which is not present in DNA (Fig.
2). Since DNA does not have the 2'-OH, different mechanisms of metal-assisted hydrolysis of RNA and DNA are
predicted.
Mutational and pH Studies of the 3' 
5' Exonuclease Activity
of Bacteriophage T4 DNA Polymerase*
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ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease activity of
proofreading DNA polymerases requires two divalent metal ions, metal
ions A and B. Mutational studies of the 3' 5' exonuclease active
center of the bacteriophage T4 DNA polymerase indicate that residue
Asp-324, which binds metal ion A, is the single most important residue
for the hydrolysis reaction. In the absence of a nonenzymatic source of
hydroxide ions, an alanine substitution for residue Asp-324 reduced
exonuclease activity 10-100-fold more than alanine substitutions for
the other metal-binding residues, Asp-112 and Asp-219. Thus,
exonuclease activity is reduced 105-fold for the
D324A-DNA polymerase compared with the wild-type enzyme, while
decreases of 103- to 104-fold are detected for
the D219A- and D112A/E114A-DNA polymerases, respectively. Our results
are consistent with the proposal that a water molecule, coordinated by
metal ion A, forms a metal-hydroxide ion that is oriented to attack the
phosphodiester bond at the site of cleavage. Residues Glu-114 and
Lys-299 may assist the reaction by lowering the pKa
of the metal ion-A coordinated water molecule, whereas residue Tyr-320
may help to reorient the DNA from the binding conformation to the
catalytically active conformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease active center where the
terminal nucleotide is removed. Mutant DNA polymerases that have
reduced ability to carry out the transfer process (1-4) or are
defective in the hydrolysis reaction (4-7) replicate DNA with more
errors (1, 5-9). A two-metal ion mechanism for the 3' 5'
exonuclease activity of Escherichia coli DNA polymerase I
(DNA pol I)1 has been
proposed from structural and mutational studies (10-12). The two
divalent metal ions, which may be Mg2+, Mn2+,
or Zn2+ (10), are bound by conserved carboxylate residues
in the exonuclease active centers of proofreading DNA polymerases
(reviewed in Refs. 13 and 14). According to the model (Fig.
1), a water molecule, coordinated by
metal ion A, forms an attacking hydroxide ion, which is positioned
in-line with the target phosphodiester bond. Metal ion B is proposed to
stabilize the leaving 3'-hydroxy group and to position the O-P-O bond
angles in the transition state (12). The two metal ions are central to
the model because amino acid substitutions that prevent binding of one
or both metal ions reduce exonuclease activity several thousandfold.
The conservation of metal ion binding residues in the exonuclease
active centers of all proofreading DNA polymerases, and the severe
reduction in exonuclease activity when the metal binding residues are
replaced by non-carboxylate residues, suggest a common mechanism for
the hydrolysis reaction catalyzed by DNA polymerases (13).
View larger version (19K):
[in a new window]
Fig. 1.
Proposed two-metal ion enzymatic mechanism
for the hydrolysis reaction catalyzed by E. coli DNA
pol I, adapted from Beese and Steitz (12).
View larger version (16K):
[in a new window]
Fig. 2.
Proposed two-metal ion model for the
hydrolysis reaction catalyzed by the hammerhead ribozyme, adapted from
Pontius et al. (16).
Structural studies of the Klenow fragment of DNA pol I suggest that a
water molecule or hydroxide ion is bound to metal ion A (Ref. 12; Fig.
1); however, only one study has examined the pH dependence of the
exonuclease reaction and correlation to metal ion
pKa values (11). We present data here from pH and buffer studies of the exonuclease reaction catalyzed by wild-type and
mutant bacteriophage T4 DNA polymerases that are consistent with
formation of a metal hydroxide ion. Our studies focus on the
ExoIII motif, which is conserved in the 3' 5'
exonuclease active centers of proofreading DNA polymerases (13, 14).
The motif sequence is a tyrosine residue, followed by three amino acids, and then an aspartate residue, which is a ligand to metal ion A
(5, 13). The conserved ExoIII residues in E. coli
DNA pol I are Tyr-497 and Asp-501 (Fig. 1). The corresponding residues in T4 DNA polymerase are Tyr-320 and Asp-324 (5, 19, 20) and Tyr-323
and Asp-327 in the T4-like RB69 DNA polymerase (Refs. 21 and 22; Fig.
3). Note the structural similarities in the exonuclease active centers
of the bacterial and phage enzymes (Ref. 22; compare Figs. 1 and 3).
The other essential residues for the 3' 5' exonuclease activity of
E. coli DNA pol I are Asp-355, which may provide another
ligand to metal ion A, and residue Asp-424, which provides a ligand to
metal ion B (Fig. 1). The corresponding phage DNA polymerase residues
are Asp-112 and Asp-219 in the T4 DNA polymerase and Asp-114 and
Asp-222 in the RB69 DNA polymerase (Fig. 3). Functional similarities
for the bacterial and phage DNA polymerases have also been
demonstrated. For example, alanine substitutions for residue Asp-501 in
E. coli DNA pol I and for residue Asp-324 in T4 DNA
polymerase reduce 3' 5' exonuclease activity by 3-4 orders of
magnitude (5-7, 11, 20). The large reductions in exonuclease activity
indicate that Asp-501 in E. coli DNA pol I and Asp-324 in T4
DNA polymerase are essential for catalysis. Smaller reductions in
exonuclease activity are detected when phenylalanine is substituted for
Tyr-497 in E. coli DNA pol I (11) and when phenylalanine or
alanine are substituted for Tyr-320 in T4 DNA pol (20); hence, the
conserved tyrosine residue in the ExoIII motif plays an
important, but ancillary role, in catalysis.
We extend previous studies (5-7, 11, 20) of mutant DNA polymerases
with reduced ability to bind metal ions in the exonuclease active
center by demonstrating that the residual exonuclease activity reported
for some of the mutants is due partially to the buffering agent, most
frequently Tris. A 10-fold lower residual exonuclease activity was
detected for the T4 D324A-DNA polymerase in HEPES compared with Tris
buffer. However, just a 2-fold lower residual activity was detected in
HEPES buffer for the D112A/E114A- and D219A-DNA polymerases. The
larger drop in residual exonuclease activity for the D324A-DNA
polymerase in HEPES compared with Tris buffer indicates that residue
Asp-324 function can be fulfilled partially by Tris buffer. We propose
that Tris may interact with the T4 DNA polymerase in the exonuclease
active center to produce a locally high concentration of hydroxide ions
or, possibly, to act directly as a nucleophile. The implication of this
proposal for the wild-type T4 DNA polymerase is that metal ion A, bound by residue Asp-324, normally provides this function by coordinating a
water molecule to form an attacking metal-hydroxide ion, as proposed
for E. coli DNA pol I (Ref. 12; Fig. 1). For T4 DNA polymerase, residues Glu-114 and Lys-299 may assist formation of the
attacking hydroxide ion at physiological pH. Additionally, T4 DNA
polymerase residue Tyr-320 appears to assist orientation of the DNA
substrate in the catalytically active conformation.
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EXPERIMENTAL PROCEDURES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Construction and Purification of T4 DNA Polymerase 3' 5'
Exonuclease Mutants--
Amino acid substitutions Y320F and D324A were
engineered into the T4 DNA polymerase expression vector (23) using
standard site-directed mutagenesis procedures (24). Mutations in the plasmid vector were verified by sequencing the entire DNA polymerase transcript (9). Wild-type and the Y320F/D324A-DNA polymerases were
purified following published methods (25). The D324A-, D112A/E114A-,
and D219A-DNA polymerases have been described (6). The enzyme
preparations were judged pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of overloaded lanes; only a single band was visible at the mobility predicted for the
103,572-dalton T4 DNA polymerase. Enzyme concentration was measured
spectrophotometrically using the experimentally determined molar
extinction coefficient at 280 nm of 1.49 × 105
M
1 cm1 (5).
DNA Polymerase Assay-- DNA polymerase activity was measured using a 60-µl volume reaction with 67 mM Tris-HCl (pH 8.8), 16.7 mM (NH4)2SO4, 0.5 mM dithiothreitol, 6.7 mM MgCl2, 167 µg/ml bovine serum albumin, 83 µM dNTPs (one labeled [3H]dNTP at about 100 cpm/pmol), and 833 µM "activated" DNA, expressed as nucleotide equivalents and prepared as described (6). The reactions were initiated by addition of wild-type or mutant T4 DNA polymerase to give a final concentration of 10 nM. A linear incorporation rate was observed for at least 30 min at 30 °C. Reactions were stopped by spotting 20 µl of the reaction mixture onto GF/A filters (Whatman) that were prespotted with 20 µl of 0.4 M EDTA. The filters were immersed in an ice-cold solution of freshly prepared 0.1 M sodium pyrophosphate and 7.5% trichloroacetic acid. The filters were washed under vacuum with cold water, dried, and counted in scintillation fluor.
3' 5' Exonuclease Assays for T4 DNA
Polymerase--
Exonuclease activity was measured by two assay
systems. One assay was similar to the conditions of the polymerase
assay described above with 67 mM Tris-HCl (pH 8.8), 16.7 mM (NH4)2SO4, 0.5 mM dithiothreitol, 6.7 mM MgCl2,
and 167 µg/ml bovine serum albumin, but the dNTPs were omitted and
the DNA substrate was 80 µM alkali-denatured, 3H-labeled E. coli DNA (100 cpm/pmol of
nucleotide) (26). Wild-type T4 DNA polymerase was present at 10 nM, but the mutant DNA polymerases were assayed at 1 µM. Reactions were stopped by the addition of 0.44 ml of
1 mg/ml unlabeled single-stranded DNA, followed by 0.5 ml of ice-cold
15% trichloroacetic acid to precipitate the undegraded substrate. The
solutions were chilled for 10 min and then centrifuged. A 200-µl
sample of the supernatant, which contains the soluble dNMPs produced by
the excision reaction, was counted in 4.8 ml of scintillation fluor.
The second exonuclease assay used p(dT)16 (Amersham
Pharmacia Biotech), which was labeled at the 5'-end by using
[
-32P]ATP and T4 polynucleotide kinase. The
exonuclease reaction mixture contained 50 mM buffer
(described below), 1 mM dithiothreitol, 7 mM
MgCl2, 12.5 nM 5' 32P-labeled
p(dT)16, and 72 µM unlabeled
p(dT)16, to give a total DNA concentration approximately
10-fold higher than the Km(app) for the
p(dT)16 substrate determined for the wild-type and
D324A-DNA polymerases (20). Tris-HCl buffer was used in the pH range
from 7.0 to 8.7 and glycine-NaOH buffer was used from pH 8.7 to 10.2. HEPES buffer, pH 8.0, was also used. Reactions were initiated by the
addition of enzyme. Wild-type T4 DNA polymerase was assayed at 4 nM, the D324A-DNA polymerase at 2 µM, and the
Y320F/D324A-DNA polymerase at 1 µM. Reactions were
incubated at 37 °C.
Reaction products were separated by running samples in 15% or 20% polyacrylamide, 8 M urea denaturing gels. Exonuclease activity was determined by measuring the populations of degradation products with a PhosphorImager (Molecular Dynamics). The gels were analyzed by the method of Cheng and Kuchta (27), which considers full-length and all partially degraded oligomers as potential substrates. Band intensities were quantitated with the ImageQuant software supplied by Molecular Dynamics (Sunnyvale, CA). The mole fraction of each species was multiplied by the number of excision events required to generate that species. For the degradation of 5' 32P-labeled p(dT)16, the molar quantity of substrate degraded is given by {[(fraction 16-mer) × 0] + [(fraction 15-mer) × 1] + [(fraction 14-mer) × 2] + ... } × (mol of DNA substrate in the assay).
3' 5' Exonuclease Assay for the Klenow Fragment of E. coli
DNA pol I--
Klenow fragment 3' 5' exonuclease activity was
examined under the assay conditions described above with the 5'
32P-end-labeled p(dT)16 substrate. The DNA
substrate was present at 20 nM and Klenow fragment
(Amersham Pharmacia Biotech) was at 75 nM.
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RESULTS |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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DNA Polymerase Activity of Mutant T4 DNA Polymerases with Reduced
3' 5' Exonuclease Activity--
Ten-fold and greater reductions in
polymerase activity were detected for the T4 D112A/E114A-, D219A-, and
D324A-DNA polymerases in previous studies (6). The reduced polymerase
activity observed for the T4 DNA polymerase exonuclease mutants
indicates interdependence between polymerase and exonuclease activities
as explained in Ref. 6. We confirmed our previous observation that DNA
polymerase activity for the D324A-DNA polymerase is reduced (Table
I). A similar or larger decrease in
polymerase activity was predicted for the doubly mutant Y320F/D324A-DNA
polymerase because the Y320F substitution also reduces polymerase
activity (20). Surprisingly, the doubly mutant Y320F/D324A-DNA
polymerase was more active in DNA polymerase assays than the
singly mutant D324A-DNA polymerase. A possible explanation for this
observation is presented under "Discussion."
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3' 5' Exonuclease Activity of Phage T4 D324A- and
Y320F/D324A-DNA Polymerases--
An alanine substitution for residue
Asp-324 reduced T4 DNA polymerase 3' 5' exonuclease activity 4 orders of magnitude (Table I), as expected since the role of Asp-324 is
in binding one of the two essential metal ions in the exonuclease
active center (Fig. 3). The low level of
exonuclease activity is due to residual rather than contaminating
exonuclease activity, as shown by neutralizing antibody studies (20).
An equal or larger reduction in exonuclease activity was expected for
the doubly mutant Y320F/D324A-DNA polymerase, because the Y320F
substitution alone reduces exonuclease activity about 100-fold (20).
The Y320F/D324A-DNA polymerase, however, was 5-fold more
active than the D324A-DNA polymerase (Table I). Thus, both higher
polymerase and 3' 5' exonuclease activities were detected for the
doubly mutant Y320F/D324A-DNA polymerase compared with the singly
mutant D324A-DNA polymerase. These observations suggest a pivotal
function for residue Tyr-320. A model for Tyr-320 function is presented
under "Discussion."
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Effect of pH on the 3' 5' Exonuclease Reaction Activity of
Wild-type and Mutant T4 DNA Polymerases--
pH titration of the 3'
5' exonuclease activity was done using 5'
32P-end-labeled p(dT)16 as the DNA substrate
(see "Experimental Procedures"). Exonucleolytic degradation of the
labeled DNA was followed by gel electrophoresis of the partially
degraded DNAs and by quantitation of the band intensities. Degradation
of the 5' 32P-end-labeled p(dT)16 substrate by
the wild-type T4 DNA polymerase is shown in Fig.
4. The use of single-stranded DNA
substrates avoids the possibility of a pH effect on strand separation.
The reactions were done at Vmax conditions with
the concentration of p(dT)16 at 72 µM, which
is approximately 10-fold higher than the Km(app)
(20). The high concentration of DNA ensures a constant population of
enzyme-DNA complexes.
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A high level of T4 DNA polymerase exonuclease activity was observed over the entire pH range examined, from pH 7.0 to 10.2, but the profile is complex, which suggests that the ionization states of several amino acid residues affect activity (Fig. 4). The pH titration profile for the exonuclease activity of T4 DNA polymerase differs from the pH titration reported for Klenow fragment in which a 45-fold stimulation in activity was observed at pH 10.2 compared with pH 7.5 (11).
The residual exonuclease activity detected for the mutant T4 D324A- and
Y320F/D324A-DNA polymerases was also examined as a function of pH
(Figs. 5 and
6, respectively). Activity was
103- to 104-fold lower for the mutant enzymes
compared with the wild-type enzyme (Table
II). Activity was not stimulated at pH
10.2. The Y320F/D324A-DNA polymerase (Fig. 6) was about 10-fold more
active than the singly mutant D324A-DNA polymerase (Fig. 5).
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Effect of Buffer on the Residual Exonuclease Activity of the
D324A-, D219A-, and D112A/E114A-DNA Polymerases--
The residual 3'
5' exonuclease activity of the mutant enzymes may be due to a low
amount of fortuitous binding of metal ions in the exonuclease active
center, despite the absence of the metal-binding aspartate ligands.
Another possibility is that reaction components, such as the buffer,
may participate in the exonuclease reaction. Tris, for example, has a
potentially reactive primary amine, and Tris may also act as a
nucleophile. Undesirable side effects of Tris have been described (28).
To investigate the possibility of a buffer effect, the exonuclease
activity of the mutant DNA polymerases was measured in the presence of
another buffer, HEPES (Fig. 7). The
exonuclease activity of the D324A-DNA polymerase was reduced more than
10-fold in HEPES compared with Tris buffer, but only a 2-fold reduction
was observed for the D219A- and D112A/E114A-DNA polymerases (Table
III). The effect of Tris on the
exonuclease activity of the D324A-DNA polymerases was proportional to
the concentration of Tris buffer present; about twice as much activity
was detected with 50 mM Tris compared with 25 mM Tris (Fig. 7).
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Effect of pH on the 3' 5' Exonuclease Reaction Catalyzed by
Klenow Fragment--
Because the 3' 5' exonuclease activity of T4
DNA polymerase was reduced at pH 10.2, rather than being strongly
stimulated as reported for Klenow fragment (11), the pH dependence of
the exonuclease reaction catalyzed by Klenow fragment was re-examined. Surprisingly, instead of the expected 45-fold stimulation in 3' 5'
exonuclease activity at pH 10.2 (11), exonuclease activity was
gradually reduced from pH 8.7 to pH 10.2 (Fig.
8). The kcat at pH
10.2 in our assay was about 0.07 s1, which is similar to
the 0.09 s1 rate reported for Klenow fragment with a
poly(dT) substrate at pH 7.5 (11). Differences in the reaction
conditions between our studies and those of Derbyshire et
al. (11) may account for the absence of a strong pH dependence at
pH 10.2 for the Klenow fragment in our reactions. Alkaline conditions
in general, however, are expected to reduce exonuclease
activity because Mg2+ is less soluble at higher pH. Thus,
exonuclease activity, which requires divalent metal ions, is expected
to decline as the concentration of Mg2+ ions is
depleted.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The Hydrolysis Reaction Catalyzed by Bacteriophage T4 DNA Polymerase-- The hydrolysis reaction in the multistep T4 DNA polymerase exonucleolytic-proofreading pathway was studied by using mutant DNA polymerases with amino acid substitutions for conserved residues in the exonuclease active center. The ExoIII motif residues, Tyr-320 and Asp-324, were changed to Y320F and D324A. The ExoI and ExoII motif residues, Asp-112/Glu-114 and Asp-219, were changed to D112A/E114A and D219A. Mutant DNA polymerases with alanine substitutions for Asp-112, Asp-219, or Asp-324 have 103- to 105-fold less exonuclease activity than the wild-type level (Refs. 5-7 and 20; Tables II and III). Studies of the residual exonuclease activity detected for the mutant DNA polymerases have revealed insights into the metal ion-dependent step(s) in the hydrolysis reaction.
Analyses of experiments with the D112A/E114A-, D219A-, D324A-, and Y320F/D324A-DNA polymerases assume that only metal ion binding and not protein structure is changed by the amino acid substitutions. Structural alterations were not detected for E. coli DNA pol I mutants with analogous substitutions (29). For T4 DNA polymerase mutants, a fluorescence assay provides information on the structural integrity of the exonuclease active center. A fluorescent pre-exonuclease complex is formed with the wild-type T4 DNA polymerase and DNA labeled with 2-aminopurine at the 3'-terminal position of the primer strand (2-4, 30). Formation of the fluorescent pre-exonuclease complex is also observed for the D324A- and Y320F/D324A-DNA polymerases, and formation is observed with the same efficiency as detected for the wild-type enzyme (30).2 Thus, the D324A substitution does not appear to impede DNA binding in the exonuclease active center. The D112A/E114A and D219A substitutions also do not affect the rate for forming fluorescent pre-exonuclease complexes, but the steady-state levels of the fluorescent complex are reduced (30). The higher residual exonuclease activities of the D112A/E114A- and D219A-DNA polymerases compared with the D324A-DNA polymerase (Table III), however, indicate that the differences detected by fluorescence intensity measurements may reflect only subtle changes in structure.
Metal ion occupancy in the exonuclease active center of the mutant T4 DNA polymerases has been predicted by comparisons to structural studies of Klenow mutants with alanine substitutions for metal binding residues (20). The metal ion B site is predicted to be vacant in the D219A-DNA polymerase, and the metal ion A site is predicted to be vacant in the D324A-DNA polymerase (Table IV). Since residue Asp-112 may provide ligands to both metal ion A and B, little or only partial metal ion occupancy is predicted at either site. This metal-binding pattern is supported by equilibrium Mg2+ binding studies (31). Two classes of Mg2+ binding sites were detected: a high affinity site, Kd about 5 µM for the wild-type T4 DNA polymerase, and a low affinity site, Kd about 2 mM. Mg2+ binding in the high affinity metal binding site, but not the low affinity site, is affected by alanine substitutions for residues Asp-112, Asp-219, and Asp-324 (31). The Mg2+ dissociation constants determined for the D219A- and D324A-DNA polymerases are similar at 11 and 27 µM, respectively, but apparent 10-fold weaker binding, Kd = 235 µM, is detected for the D112A/E114A-DNA polymerase. These data are consistent with a single metal ion bound in the exonuclease active centers of the D219A- and D324A-DNA polymerases and a severe reduction in binding of both metal ions by the D112A/E114A-DNA polymerase.
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The first step in the proposed two-metal ion model for the DNA polymerase hydrolysis reaction is formation of an attacking metal hydroxide ion by metal ion A (Fig. 1). If residues that bind metal ion A are missing, then any residual exonuclease activity is expected to derive from hydroxide ions produced by interactions between water molecules and between water molecules and the buffer. The contribution of buffer to the residual exonuclease activity was assessed by using HEPES in place of the potentially reactive and interactive Tris (Fig. 7, Table III). The residual exonuclease activity of the D324A-DNA polymerase was reduced more than 10-fold in HEPES compared with Tris buffer, and 2-fold reductions were detected for the D112A/E114A- and D219A-DNA polymerases. The stronger buffer effect observed for the D324A-DNA polymerase indicates a role for residue Asp-324 and metal ion A in producing hydroxide ions.
The role of residue Asp-324 and metal ion A in producing hydroxide ions is supported by studies of the residual exonuclease activity of the D219A- and D112A/E114A-DNA polymerases. The highest level of exonuclease activity was observed for the D219A-DNA polymerase (Table III). This mutant is predicted to retain metal ion binding in site A and, thus, according to the model, still has the potential to generate an attacking metal-hydroxide ion. If production of a metal-hydroxide ion is rate-limiting, then this mutant is expected to have higher activity, which is observed.
Residue Asp-112 is also predicted to provide a ligand to metal ion A, but some metal binding in site A may be possible in the D112A/E114A-DNA polymerase due to residue Asp-324 (Table IV). No metal A binding is predicted, however, for the D112A/D324A-DNA polymerase, which has lost both carboxylate ligands to metal ion A. In keeping with this proposal, 10-fold less residual activity is detected for the D112A/D324A-DNA polymerase compared with the D112A/D219A- or D219A/D324A-DNA polymerase (20). The D112A/D219A- and D219A/D324A-DNA polymerases also have the same amount of residual exonuclease as the singly mutant D112A- and D324A-DNA polymerases. Thus, the largest reduction in hydrolysis activity is observed only when both ligands to metal ion A, Asp-112 and Asp-324, are removed. These experiments (20) were done in Tris buffer; hence, a larger reduction in residual exonuclease activity is predicted for the D112A/D324A-DNA polymerase in HEPES buffer. The proposed partial occupancy of metal site A caused by the D112A substitution differs from structural studies of the D355A/E357A-Klenow fragment in which no metal binding was observed in metal sites A or B (Fig. 1; Ref. 28). Structural studies, however, may not have sufficient sensitivity to detect partial or transient metal ion binding.
Additional residues in the exonuclease active center of the T4 DNA
polymerase appear to assist the hydrolysis reaction. A comparison to
carboxypeptidase A, a zinc metalloenzyme, is informative (32). A
bell-shaped pH-rate profile is observed for the peptide bond cleaving
reaction catalyzed by carboxypeptidase A with inflection points at pH
6.1 and 9.0, which suggests the importance of both acidic and basic
catalysis. Hydrogen bonding between a zinc-coordinated water molecule
and a nearby glutamate residue in the active center is proposed to
reduce the pKa of the coordinated water from pH 10 to 9 (31, 32). We propose a similar model for generation of hydroxide
ions in the exonuclease active center of T4 DNA polymerase (Fig.
9). The metal ion bound in site A in the
T4 DNA polymerase exonuclease active center may also be
Zn2+ (12, 13, 19). Mutational analysis implicates
important, but auxiliary roles for residues Glu-114 and Lys-299 in the
T4 DNA polymerase exonuclease active center and the corresponding residues Glu-116 and Lys-302 in the RB69 DNA polymerase (Fig. 3; Ref.
20). Although alanine substitutions in the T4 DNA polymerase for
Glu-114 or Lys-299 reduce exonuclease activity just 5-fold (20),
reactions were done in Tris buffer, which provides a possible nonenzymatic source of hydroxide ions, as discussed above. Thus, larger
reductions in exonuclease activity may be observed for the E114A- and
K299A-DNA polymerases in HEPES buffer. Residue Glu-114 in the T4 DNA
polymerase (Glu-116 in the RB69 DNA polymerase or Glu-357 in DNA pol I)
may lower the pKa of a coordinated water molecule as
proposed for carboxypeptidase A. In addition, structural studies of the
T4-like, RB69 DNA polymerase show that the 
-amino group of Lys-302
is directed toward the scissile phosphate and is in position to
interact with the metal-A binding residue Asp-327 (Ref. 22; Fig. 3). An
ionized -amino group may interact with one of the oxygens of the
carboxylate group of Asp-324 in the T4 DNA polymerase (Asp-327 in the
RB69 DNA polymerase), which would increase the electronegativity of the
remaining oxygen (Fig. 9). Thus, the combined interactions of Glu-114
with the coordinated water molecule and interactions between Lys-299
with Asp-324 and the bound metal ion may reduce the
pKa of the coordinated water molecule. These
interactions, and perhaps additional interactions in the exonuclease
active center, may assist formation of the attacking metal-hydroxide
ion at physiological pH.
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The model for formation of a metal hydroxide ion by T4 DNA polymerase requires both oxygens of Asp-324 (Fig. 9). Mutational analysis indicates that both oxygens are required for exonuclease activity of the T4 DNA polymerase (20), but not for E. coli DNA pol I (11). DNA pol I also lacks a lysine residue that corresponds to T4 Lys-299 or RB69 Lys-302 (22). These differences could provide part of the explanation for why the T4 DNA polymerase has about 1000-fold more exonuclease activity than DNA pol I (33). The proposed role for Lys-299 in the T4 DNA polymerase to increase the electronegativity of the metal-binding carboxylate oxygen of residue Asp-324 is also consistent with Asp-324 being the single most important residue in the hydrolysis reaction catalyzed by the T4 DNA polymerase.
Residue Tyr-320 in T4 DNA Polymerase Affects DNA Binding in the
Exonuclease Active Center--
Residues Tyr-320 in T4 DNA polymerase
and Tyr-497 in Klenow fragment appear to play secondary roles in the
exonuclease reaction since alanine or phenylalanine substitutions for
these residues reduce exonuclease activity less than alanine
substitutions for any of the conserved aspartate residues required for
metal binding (11, 20). Structural studies of Klenow fragment and the
T4-like RB69 DNA polymerase suggest that the phenolic side chain of the conserved tyrosine is in position in the transition state to orient the
attacking water molecule or hydroxide ion (Fig. 1; Refs. 12 and 22).
Thus, we anticipated that the doubly mutant Y320F/D324A-T4 DNA
polymerase would have similar or less exonuclease activity than the
singly mutant D324A-DNA polymerase. Surprisingly, the 3' 5'
exonuclease activity of the Y320F/D324A-DNA polymerase was 10-fold more
active than the exonuclease activity of the D324A-DNA polymerase (Table
II, Figs. 5 and 6).
The phenolic group of the conserved tyrosine could reduce activity by inhibiting catalysis or by reducing DNA binding in the exonuclease active center. Experiments that measured partitioning of DNA between the polymerase and exonuclease active centers of Klenow fragment found that Tyr-497, which corresponds to residue Tyr-320 in the T4 DNA polymerase, affects DNA binding in the exonuclease active center (35). Increased partitioning of DNA to the exonuclease active center was observed for the Y497A/D424A-Klenow mutant compared with the D424A-Klenow mutant. To reconcile the seemingly contradictory observations that an alanine substitution for Tyr-497 in Klenow fragment can both reduce exonuclease activity, but increase partitioning of DNA to the exonuclease active center, Lam et al. (35) suggest that Tyr-497 may affect different binding conformations in the exonuclease active center. Thus, residue Tyr-497 in Klenow may interfere with the initial binding mode, but may assist in reorienting the DNA substrate to a catalytically optimal conformation. The T4 D324A- and Y320F/D324A-DNA polymerases, however, form the fluorescent pre-exonuclease complex with apparent equal efficiency.2 Thus, residue Tyr-320 must affect a reaction step that follows formation of the pre-exonuclease complex, but before hydrolysis in order to explain the higher exonuclease activity of the T4 Y320F/D324A-DNA polymerase compared with the D324A-DNA polymerase.
Interdependence of Polymerase and 3' 5' Exonuclease Activities
of T4 DNA Polymerase--
Unlike Klenow fragment, many amino acid
substitutions in the T4 DNA polymerase that reduce exonuclease activity
also reduce polymerase activity (6, 20). Although the reductions in
polymerase activity are small compared with the reductions in
exonuclease activity (Table I), the reduced polymerase activity
demonstrates the interdependence between reactions catalyzed in the
polymerase and exonuclease active centers. Such interdependence is
expected for a DNA polymerase, like T4 DNA polymerase, which alternates between polymerase and exonuclease activities without dissociating from
the DNA primer-template (36). One explanation for the decrease in
polymerase activity detected for the exonuclease-deficient mutants is
that reduced ability to bind DNA in the exonuclease active center may
result in dissociation. Amino acid substitutions that can restore
processivity by assisting DNA binding, as suggested for the Y320F
substitution, can increase both the polymerase and exonuclease
activities of the D324A-DNA polymerase (Table I).
| |
ACKNOWLEDGEMENTS |
|---|
We thank M. Otto for the initial observation
that the 3' 5' exonuclease activity of the D324A-DNA polymerase is
reduced significantly in HEPES buffer; L. Bloom for preliminary
experiments; J. Wang, and T. Steitz for providing Fig. 3; B. Poulin for
assistance in formatting Fig. 3; and C. Joyce, W. Konigsberg, W. Lott,
and E. Fidalgo da Silva for detailed comments on the manuscript.
Technical assistance was provided by R. Nonay and Z. Ozum.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, Collaborative Research Grant Program CPG0163278, and Medical Research Council of Canada Grant MT-14300 (to L. J. R.-K.).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.
Present address: Dept. of Medical Biophysics, University of
Toronto, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada.
§ Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 403-492-5383; Fax: 403-492-9234; E-mail: lreha@gpu.srv.ualberta.ca.
2 E. Elisseeva, S. Mandal, and L. Reha-Krantz, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: DNA pol I, E. coli DNA polymerase I; p(dT)16, poly(dT)16.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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), and Gly-NaOH
buffer is indicated by triangles (
). Degradation rates
are normalized to picomoles of substrate degraded per 20 pmol of enzyme
in order to allow ready comparison of the wild-type T4 DNA polymerase
to the exonuclease-deficient T4 DNA polymerases. The pH values in
panel B are positioned below the corresponding
gel lanes in panel A.
" symbol indicates that the metal site is expected to be vacant.
The "+/