 |
INTRODUCTION |
When dsDNA1 is
replicated, repaired, or recombined, the necessary ssDNA intermediates
are provided by the activity of helicases (1-5). These enzymes appear
to be ubiquitous, having been identified in viral, bacterial, and
eukaryotic systems. Helicases hydrolyze nucleotide triphosphates,
usually ATP, to obtain the energy needed to unwind dsDNA. They
translocate on DNA often in a very processive manner, unwinding
thousands of base pairs in a single binding event. Their processive
nature implies that helicases have multiple DNA binding sites; indeed,
a characteristic feature of many helicases is their propensity to form
oligomeric structures, often dimers or hexamers.
Knowledge of the oligomeric form of a helicase is of fundamental
concern in development of a mechanism for unwinding activity, and many
approaches have been applied to determine oligomeric structure. The
lack of evidence for oligomerization from biophysical experiments has
led to the suggestion that some helicases function as monomers. PcrA
helicase of Bacillus stearothermophilus is proposed to
function as a monomer that contains two binding sites for DNA (6, 7).
Evidence has been provided that suggests Escherichia coli UvrD helicase (helicase II) can be active as a monomer
in vivo and in vitro (8). The E. coli
Rep helicase has been proposed to function as a dimer (2), whereas
other helicases such as T7 gene 4 helicase and T4 gp41 helicase appear
to function as hexamers (9, 10).
Helicases have been classified according to sequence homology (11), and
those enzymes in superfamily 1 and superfamily 2 have proven difficult
to characterize in terms of oligomerization. The focus of this study is
the bacteriophage T4 helicase, Dda, a 5'-to-3' helicase classified in
superfamily 1. Evidence suggests that Dda is involved in T4 replication
initiation (12). T4 Dda
mutants show a delayed DNA
synthesis phenotype, consistent with this hypothesis. Dda also enhances
the rate of branch migration owing to a specific interaction with the
T4 recombinase (UvsX) and is therefore likely to play a role in
recombination (13-15). Dda binds tightly to the T4 single-stranded
DNA-binding protein, gp32, although the significance of this
interaction has not been determined (15, 16).
Dda translocates on ssDNA with a strong directional bias in the
5'-to-3' direction (17). It is capable of removing protein blocks
placed in the path of the enzyme, including streptavidin bound to
biotin-labeled oligonucleotides (18). Dda is not highly processive (19,
20). Unwinding of only a few hundred base pairs can be prevented by
addition of ssDNA, even when Dda is incubated with the substrate prior
to addition of the competitor DNA (19). In contrast, the replicative
helicase from bacteriophage T4, gp41, can unwind thousands of base
pairs in a single binding event (21). Like T7 gene 4 helicase, gp41 is
a hexameric helicase that encircles ssDNA, resulting in very high
processivity (18, 22). The oligomeric nature of Dda was investigated in
order to determine whether its relatively low processivity might be related to the oligomeric structure, and to lay the foundation for
future mechanistic studies.
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EXPERIMENTAL PROCEDURES |
Reagents--
Phosphoenolpyruvate kinase/lactate dehydrogenase,
NADH, pepstatin A, phenylmethylsulfonyl fluoride, and lysozyme were
from Sigma. ATP, Hepes, glycerol, EDTA, NaCl,
isopropyl-1-thio-
-D-galactopyranoside, dextrose, and
KOAc were from Fisher. T4 polynucleotide kinase was purchased from New
England Biolabs. [
-32P] ATP was purchased from
PerkinElmer Life Sciences. DSP was purchased from Pierce. All
oligonucleotides were purchased from Operon Technologies. Oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis on a SE 410 Sturdier Vertical Slab Unit (Amersham Pharmacia Biotech). Oligonucleotides were visualized by UV shadowing, and the appropriate band was excised. DNA was removed from the acrylamide by electroelution by using an Elutrap apparatus (Schleicher & Schuell). Eluted DNA was loaded onto a Waters C18 Sep-Pak
cartridge, de-salted with H2O, then eluted with 60%
methanol. DNA was dried in a Speed Vac and then redissolved in 10 mM Hepes (pH 7.5) and 1 mM EDTA. DNA was
quantified by UV absorbance at 260 nm in 0.2 M KOH, and the
concentration was determined by using a calculated extinction coefficient.
WT Dda Expression and Purification--
Recombinant
plasmid pET26b-Dda was kindly provided by Dr. Craig Cameron
(Pennsylvania State University). Dda was expressed in E. coli BL21/DE3 cells. Expression was induced at 15 °C overnight by addition of 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside and 0.2%
dextrose. Cells (30 g) were collected by centrifugation then suspended
in 175 ml of lysis buffer (25 mM Tris acetate (pH 7.6), 500 mM NaCl, 1 mM EDTA, 5 mM BME, 10%
glycerol, 2 mM phenylmethylsulfonyl fluoride, 4 µg/ml
pepstatin A, and 0.2 mg/ml lysozyme). Cell lysis was assisted by one
cycle of freezing and thawing followed by sonication. The lysate was
cleared by centrifugation at 20,000 rpm in a JA-25.50 rotor (Beckman)
for 20 min at 4 °C. The supernatant was further clarified by
centrifugation for 2 h at 45,000 rpm in a 50.2 TI rotor (Beckman)
at 4 °C. The supernatant was loaded onto a 30-ml Macro Prep High Q
anion exchange column (Bio-Rad) equilibrated with lysis buffer. The
proteins in the flow-through fraction from this column were
precipitated with 55% ammonium sulfate, and the pellet was collected
by centrifugation at 20,000 rpm on a JA-25.50 rotor (Beckman) for
1 h at 4 °C. The pellet was suspended in buffer A (25 mM Tris acetate (pH 7.6), 50 mM NaCl, 1 mM EDTA, 5 mM BME, and 10% glycerol)
containing 1.5 M ammonium sulfate. Protein was loaded onto
a 40-ml methyl-HIC column (Bio-Rad) and eluted with a linear gradient
of 1.5-0 M ammonium sulfate in buffer A. Fractions
containing Dda eluted from the column from 900-600 mM
ammonium sulfate as determined by SDS-PAGE. Fractions were dialyzed
into buffer A and loaded onto a 20-ml ssDNA cellulose column (Sigma).
Dda eluted from 0.9 M to 1.5 M NaCl using a
linear gradient from 50 mM to 2 M NaCl in
buffer A. Fractions containing Dda were pooled, concentrated to ~15
ml by ultrafiltration through a 10,000 molecular weight limit filter (Amicon, Millipore), and dialyzed into buffer A. Protein was loaded onto a 15-ml Macro Prep High Q anion exchange column equilibrated in
buffer A. Dda eluted from 0.25 M to 0.32 M NaCl
using a linear gradient from 50 to 500 mM NaCl as
determined by SDS-PAGE analysis. Dda fractions were concentrated to
~3 ml using an Amicon concentrator and then dialyzed into storage
buffer (25 mM Hepes (pH 7.5), 50 mM NaCl, 2 mM BME, 1 mM EDTA, and 20% glycerol). Some Dda
was dialyzed into storage buffer in the absence of BME for chemical cross-linking experiments. Protein concentration was measured by UV
absorbance at 280 nm. A calculated extinction coefficient (59,010 cm
1·M
1)
based on the amino acid sequence was used to quantify the protein (23).
Aliquots were frozen in liquid N2 prior to storing at
80 °C. Approximately 7 mg of purified protein was obtained from 30 g of cell paste.
Expression and Purification of Mutant (K38A) Dda--
A lysine
residue at amino acid position 38 (AAG) was mutated to alanine (GCG).
The mutation was introduced using the Quick-Change kit for
site-directed mutagenesis from Stratagene and confirmed by DNA
sequencing. K38A Dda was expressed in E. coli BL21/DE3 cells
as described above for the wild type enzyme and purified by a slight
modification of the above procedure. The concentration of NaCl in the
lysis buffer was reduced to 200 mM. The cell lysate was
loaded directly onto a ssDNA cellulose column and eluted as described
above. K38A Dda eluted from the ssDNA cellulose column from 0.87 M to 1.1 M NaCl. Fractions containing K38A Dda
were identified by SDS-PAGE, collected, and dialyzed into buffer A, followed by anion exchange chromatography as described above. K38A Dda
eluted from the anion exchange column from 0.05 M to 0.25 M NaCl. The fractions containing K38A Dda were dialyzed
into storage buffer (25 mM Hepes (pH 7.5), 50 mM NaCl, 2 mM BME, 1 mM EDTA, and
20% glycerol) and quantitated by A280, using an
extinction coefficient of 59,010 cm
1·M
1.
Aliquots were frozen in liquid N2 and stored at
80 °C.
20 mg of purified K38A Dda was obtained from 10 g of cell paste.
Protein-Protein Cross-linking--
DSP was dissolved in
Me2SO up to concentrations of 100 and 50 mM.
Dda (3 µM, stored in the absence of BME) was incubated in buffer (25 mM Hepes, pH 8.2) for 5 min prior to addition of
other components such as 10 mM Mg(OAc)2, 1.1 µM 30-mer oligonucleotide, or 2 mM ATP. After
addition of the other components, incubation continued for 1 min after
which the cross-linking agent (2 or 4 mM DSP) was added to
the mixture. The cross-linking reaction proceeded for 3 min followed by
addition of glycine (1.0 M) to quench the reaction. Samples
were then analyzed by SDS-PAGE on a 4-20% pre-cast gradient gel
(Jule). Protein was visualized by silver staining.
High Pressure Gel Filtration Chromatography--
The oligomeric
nature of Dda was examined by high pressure gel filtration
chromatography by using a Bio-Select SEC 250-5 column (Bio-Rad). A
flow cell was placed into an SLM Aminco-Bowman fluorescence spectrometer so that elution from the column could be monitored by
fluorescence of the protein with an excitation wavelength of 280 nm and
an emission wavelength of 340 nm. The proteins that were used to
prepare a calibration curve were: thyroglobulin (bovine), 670 kDa;
-globulin (bovine), 443 kDa; apoferritin, 158 kDa; bovine serum
albumin, 66 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa. Gel filtration
chromatography was performed using a standard elution buffer (50 mM Tris-Cl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA) with 1 mM ATP and 1 mM
Mg(OAc)2. 3 µM Dda was incubated in the
elution buffer with ATP and MgCl2 for 2 min prior to
injection onto the column.
Protein-DNA Binding Assay--
Binding of Dda to DNA was
analyzed by fluorescence polarization using a Beacon fluorescence
polarization spectrophotometer (PanVera). A 30-mer oligonucleotide was
labeled with fluorescein on the 5' end during DNA synthesis (IDT). The
30-mer and a complementary 15-mer were purified by denaturing
polyacrylamide gel electrophoresis (see Fig. 4 for sequences). A 1:2
mixture of the 30-mer and 15-mer, respectively, was heated to 95 °C,
then slow-cooled to form a 30:15-mer partial duplex, which was purified
by native polyacrylamide gel electrophoresis. Varying amounts of Dda or
K38A Dda were added to a 1-ml aliquot of binding buffer (25 mM Hepes (pH 7.5), 0.1 mM EDTA, 150 mM KOAc, 0.1 mg/ml bovine serum albumin, and 2 mM BME) containing 1 nM of the
fluorescein-labeled 30:15-mer. Each sample was allowed to equilibrate
in solution for 5 min, after which fluorescence polarization was
measured. A second reading was taken after 30 min, in order to ensure
that the mixture had equilibrated. Less than 5% change was observed
between the 30-min measurement and the 5-min measurement indicating
that equilibrium was reached. ATP
S (0.7 mM) and
Mg(OAc)2 (10 mM) were then added to the
samples, and polarization was measured after 5 min and again after 30 min. Measurements varied by less than 5% between the 30-min and 5-min
measurements. The equilibrium dissociation constant was determined by
plotting polarization as a function of protein concentration and
fitting the data to the equation for a hyperbola by using the program
KaleidaGraph (Synergy Software).
ATPase Assays--
ATPase activity was determined using a
coupled spectrophotometric assay (24). The reaction mixture contained
25 mM Hepes (pH 7.5), 150 mM KOAc, 10 mM Mg(OAc)2, 5 mM ATP, 4 mM phosphoenolpyruvate, 21.6 units/ml phosphoenolpyruvate
kinase, 33.2 units/ml lactate dehydrogenase, 0.9 mM NADH, 2 mM BME, and ssDNA. The source for ssDNA was either poly(dT)
or denatured salmon testes (st) DNA. Final volume for each reaction
mixture was 700 µl. ATP hydrolysis rates were determined by measuring
the conversion of NADH to NAD+ at 380 nm. The extinction
coefficient of NADH at this wavelength is 1210 M
1 cm
1.
The oxidation of 1 mol of NADH corresponds to the hydrolysis of 1 mol
of ATP.
DNA Unwinding by Dda under Conditions of Excess Enzyme--
The
partial duplex DNA substrate consisted of a 30-mer DNA oligonucleotide
(the "loading" strand) annealed to a 15-mer DNA oligonucleotide
(the "displaced" strand). This substrate was created by mixing 20 pmol of radiolabeled loading strand (30-mer) with 20 pmol of displaced
strand (15-mer), passing through two Sephadex G-25 spin columns,
heating to 95 °C for 10 min, and slow cooling. Unwinding reactions
were performed using a Kintek rapid chemical quench-flow instrument.
All concentrations are after initiating the reaction. 250 nM WT Dda was incubated with 2 nM substrate at
25 °C in unwinding buffer (25 mM Hepes (pH 7.5), 150 mM KOAc, 0.1 mg/ml bovine serum albumin, 2 mM
BME, 0.1 mM EDTA). In separate experiments, varying
concentrations of K38A Dda were incubated with WT Dda in unwinding
buffer for 3 min at 25 °C, followed by addition of 2 nM
substrate. After incubation for at least 5 min, the reaction was
initiated by rapid mixing of the DNA-protein complex with 10 mM Mg(OAc)2 and 5 mM ATP in
unwinding buffer. In order to observe the first cycle of the unwinding
reaction, a protein trap (dT15, 5 µM) was
added at the same time as the ATP and Mg(OAc)2. The
unwinding reaction was allowed to proceed for varying times ranging
from 0.002 to 0.6 s, after which it was stopped by rapid addition
of quench solution (400 mM EDTA (pH 8.0)). In order to
prevent reannealing of the ssDNA products, a trapping strand that was
complementary to the displaced strand of the duplex was included in the
receiving vial at a concentration 30-fold above that of the substrate.
An aliquot (20 µl) of each sample was mixed with gel loading buffer
(0.1% xylene cyanol, 0.1% bromphenol blue, and 10% glycerol) for
analysis by native 15% polyacrylamide gel electrophoresis. The
fraction of duplex substrate and ssDNA product in each sample was
determined using a Molecular Dynamics 445-SI PhosphorImager with
ImageQuant software. The fraction of product was determined as
described (25).
DNA Unwinding under Conditions of Excess DNA Substrate--
20
pmol of freshly radiolabeled loading strand (30-mer) was mixed with 20 pmol of displaced strand (15-mer), passed through two Sephadex G-25
spin columns, and heated to 95 °C, followed by slow cooling to room
temperature. The radiolabeled substrate was added to a concentrated
stock solution (10 µM) of unlabeled 30:15-mer. The
resulting solution was used for steady-state unwinding experiments. All
concentrations are after initiating the reaction. WT Dda (5 nM) and various concentrations of mutant Dda ranging from
10 to 40 nM, were incubated for 2 min at 25 °C in
unwinding buffer plus an ATP regenerating system which was made up of 4 mM phosphoenolpyruvate, 21.6 units/ml phosphoenolpyruvate
kinase, and 33.2 units/ml lactate dehydrogenase. 400 nM
30:15-mer DNA substrate was then added to the mixture of proteins,
followed by another incubation period of 5 min. The unwinding reaction was initiated by adding 5 mM ATP, 10 mM
Mg(OAc)2, and a final concentration of 6 µM
trapping strand (the 15-mer complementary to the displaced strand) in
unwinding buffer. At various times ranging from 10 to 60 s,
10-µl aliquots were removed from the reaction and added to 10 µl of
quench solution (200 mM EDTA (pH 8.0) and 0.7% SDS).
Samples were analyzed by native 15% polyacrylamide electrophoresis.
The fraction of substrate and product in each sample was
determined by using a Molecular Dynamics 445-SI PhosphorImager with ImageQuant software, and data were analyzed as described (25,
26).
 |
RESULTS |
Protein-Protein Cross-linking--
The oligomeric nature of many
different helicases has been studied using protein-protein
cross-linking. In many cases, ligands such as ATP, Mg2+, or
DNA induce oligomerization. In the presence of a variety of ligands,
chemical cross-linking with DSP revealed only a small amount of dimeric
protein under all conditions tested (Fig.
1). Longer reaction times gave similar
results, as did other cross-linking agents such as dimethyl
suberimidate or bis(sulfosuccinimidylsuberate) (data not shown). Under
similar conditions in the presence of ATP, gp41 helicase is readily
cross-linked by DSP, giving rise to hexamers and larger species (10,
18). The results from cross-linking of Dda may be due to nonspecific
protein-protein interactions of a monomeric species or may indicate
weak, protein-protein interactions.

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Fig. 1.
Chemical cross-linking of Dda with DSP.
Dda (3 µM) was incubated in Hepes buffer (25 mM, pH 8.2) with the components listed above for 1 min at
37 °C. DSP dissolved in Me2SO was added to the mixture
and incubated for 3 min at 37 °C, after which the reaction was
quenched by addition of glycine (1.0 M). Final
concentration of DSP was 2 mM (lanes
2-5) or 4 mM (lanes
6-9). Final concentrations of ATP and Mg(OAc)2
were 2 and 10 mM, respectively. Final concentration of the
30-mer oligonucleotide was 1.1 µM. Samples were analyzed
by SDS-PAGE on a 4-20% gradient pre-cast gel (Jule, Inc.), and
visualized by silver staining.
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High Pressure Gel Filtration Chromatography--
A Bio-Select
column (Bio-Rad) was utilized for gel filtration chromatography
studies. Dda elutes from this column corresponding to a molecular mass
of ~ 28 kDa, although the actual molecular mass of Dda is ~50
kDa (Fig. 2). The late elution time
indicates that Dda may interact with the column material.
Alternatively, the molecular shape of Dda may allow the protein to
occupy smaller volumes within the column matrix than would be expected
for a 50-kDa protein. In either case, the results from these studies are inconclusive because of the unusual retention time of Dda. Other
groups have reported the unusual behavior of Dda on gel filtration
columns (19).

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Fig. 2.
High pressure gel filtration chromatography
of Dda. Gel filtration chromatography was performed using a
standard elution buffer (50 mM Tris-Cl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA) with 1 mM ATP
and 1 mM Mg(OAc)2. 3 µM Dda was
incubated in the elution buffer with ATP and MgCl2 for 2 min prior to injection onto the column. The elution of Dda was
monitored by intrinsic protein fluorescence and is shown overlaid with
the calibration plot ( ). The apparent molecular mass of Dda was 28 kDa under these conditions.
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Dda Does Not Exhibit Concentration-dependent ATPase
Activity--
The ssDNA-stimulated ATPase activity of several
helicases has been studied as a function of enzyme concentration. In
the case of UvrD and NS3h (24, 27), an increase in specific activity at
increasing protein concentration provided evidence for oligomerization. Dda was incubated in assay buffer along with 20 µM
poly(dT) (nt), which is at least 10-fold greater than the apparent
KD which is ~1-2 µM (17). Little
change in the specific activity for ATP hydrolysis was observed from 4 to 100 nM Dda (Fig. 3). The
lack of a change does not preclude formation of oligomeric species, it
simply indicates that if Dda self-assembles, the monomeric units
exhibit ATPase activity that is independent of one another under these
conditions.

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Fig. 3.
DNA-stimulated ATPase activity of Dda at
varying enzyme concentrations. ATPase activity was measured using
a coupled spectrophotometric assay. Helicase was incubated in ATPase
reaction buffer, and ATPase activity was quantitated by observing the
change in A380 over time as NADH is oxidized to
NAD+ through the coupled assay. Varying concentrations of
Dda were mixed with DNA (poly(dT), 20 µM nt), 5 mM ATP, and 10 mM Mg(OAc)2 at
25 °C. Specific activity ( ) is plotted as a function of enzyme
concentration.
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The ATPase-deficient Mutant Dda (K38A) Is Capable of Binding
DNA--
One of the conserved sequences among all members of the
various helicase families is a nucleotide-binding loop of the amino acid sequence (G/A)XXGXGK(T/S) (11). Mutations in
this region have been made in several helicases to create ATPase
deficient mutants that were incapable of unwinding duplex DNA
substrates, but were still able to form oligomers and/or bind to ssDNA.
For example, the first X residue of the conserved sequence
was mutated from arginine to cysteine in the E. coli DnaB
helicase (28), and the lysine residue was mutated to alanine in the T7
bacteriophage gene 4A helicase (29). In Dda, we have used site-directed
mutagenesis to change the DNA sequence corresponding to amino acid 38 from AAG to GCG, which converts the lysine residue in the sequence GPAGTGKT to an alanine (Fig.
4A). A series of experiments
have been performed to determine the effect of this K38A Dda mutant protein on the activity of WT Dda.

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Fig. 4.
Preparation of ATPase-deficient Dda and
equilibrium binding to DNA. A, sequence of motif I for
SF1 helicases. The consensus sequence is shown along with those
residues that display moderate variation. The lysine residue in
bold was mutated to an alanine. B, a 30-mer
containing a 5'-fluorescein label was hybridized to a 15-mer to prepare
the helicase substrate. C, binding of Dda to the substrate
was determined by measuring the change in fluorescence polarization of
the fluorescein by using a Beacon Fluorescence spectrometer (PanVera).
Varying amounts of Dda or K38A Dda were added to binding assay buffer
containing 1 nM 30:15-mer. Fluorescence polarization was
measured and data were fit to a hyperbola (KaleidaGraph). The WT enzyme
( ) bound to the 30:15-mer with a KD of 31 ± 1 nM, whereas K38A Dda ( ) bound with a
KD of 14 ± 1 nM. In the presence
of 0.7 mM ATP S and 10 mM Mg2+,
WT Dda ( ) bound with a KD of 318 ± 26 nM and the K38A Dda ( ) bound with a
KD of 107 ± 6 nM.
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Equilibrium binding assays were performed to ensure that the K38A
mutation did not compromise the ability of the mutant Dda to bind DNA.
A 30:15-mer oligonucleotide (Fig. 4B) containing a
fluorescein label on the 5'-end of the 30-mer was titrated with either
WT Dda or K38A Dda, and the resulting change in fluorescence polarization was measured (Fig. 4C). The WT enzyme bound to
the 30:15-mer with a KD of 31 ± 1 nM, whereas K38A Dda had a KD of 14 ± 1 nM (Fig. 4C). In the presence of 0.7 mM ATP
S and 10 mM
Mg2+, WT Dda bound with a KD
of 318 ± 26 nM and the mutant Dda bound with a
KD of 107 ± 6 nM (Fig.
4C). Thus, the single mutation resulted in only a small
affect on the affinity of K38A Dda for the 30:15-mer relative to WT Dda.
The Presence of Mutant Dda Does Not Decrease the Rate of ATP
Hydrolysis by WT Dda--
After generating the Dda mutant, its effect
on WT Dda ATP hydrolysis activity was studied using the coupled
spectrophotometric ATPase assay. 20 nM WT Dda did not
hydrolyze ATP to a measurable extent in the absence of stDNA, but had a
hydrolysis rate of 1652 nM·s
1
in the presence of 0.2 mM stDNA (concentration in
nucleotides). 20 nM K38A Dda was incapable of hydrolyzing
ATP in either the absence or presence of stDNA (Fig.
5A). Various ratios of WT Dda and K38A Dda protein were mixed to determine the effect of the mutant
on WT Dda ATP hydrolysis. In each experiment, the total protein
concentration (WT + K38A) was held constant at 20 nM. The
ATPase activity of 10 nM WT Dda is shown in Fig.
5A. 10 nM WT Dda hydrolyzed ATP at a rate of 705 nM·s
1 in the presence of stDNA,
while in the absence of DNA no ATP hydrolysis was observed. In the
presence of 10 nM K38A Dda, 10 nM WT Dda
hydrolyzed ATP at a rate of 826 nM·s
1. Thus, addition of the
K38A Dda did not substantially change the rate of ATP hydrolysis by the
WT enzyme (Fig. 5B). This outcome supports the previous
result (Fig. 3) and suggests that, if Dda forms oligomers, the ATPase
activity of each subunit is independent of oligomerization.

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Fig. 5.
ATPase assay of Dda in the presence of K38A
Dda. A, ATPase activity 20 nM K38A Dda
( ), 20 nM K38A Dda + stDNA ( ), 10 nM WT
Dda ( ), 10 nM WT Dda + st DNA ( ), 10 nM
WT Dda + 10 nM K38A Dda ( ), and 10 nM WT Dda + 10 nM K38A Dda + st DNA ( ). B, effect of
mutant Dda on WT Dda ATPase activity. WT Dda was mixed with K38A Dda
such that the total protein concentration was 20 nM. The
specific activity is plotted on the y axis, and the
concentration of Dda is plotted on the x axis. The
concentration of K38A Dda is plotted on the x axis in
parentheses.
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Unwinding by Dda in the Presence of K38A Dda under Conditions of
Excess Enzyme--
Unwinding experiments were performed using a Kintek
rapid chemical quench-flow apparatus to investigate the effect of K38A Dda on DNA unwinding by WT Dda. A 30:15-mer substrate (2 nM) was incubated with 250 nM WT Dda and
varying concentrations of K38A Dda. The unwinding reaction was
initiated by addition of 5 mM ATP, 10 mM
Mg(OAc)2, and 5 µM oligo(dT)15.
At varying times, the reaction was quenched by addition of 400 mM EDTA. Samples were analyzed by native polyacrylamide gel
electrophoresis (Fig. 6A), and
the quantity of ssDNA formed over time was determined by using a
PhosphorImager and ImageQuant software (Molecular Dynamics). Under
these conditions, helicase that dissociates from the substrate is
trapped by the oligo(dT)15, thereby allowing only the first cycle of unwinding to be observed during the short time frame. The
fraction of substrate unwound during the single cycle is shown over
time (Fig. 6B). Dda unwound the substrate with a pseudo
first-order rate of 24 ± 5 s
1 and an
amplitude of 0.44 ± 0.04 (Fig. 6B), whereas K38A Dda
did not unwind the duplex (data not shown).

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Fig. 6.
DNA unwinding by WT Dda in the presence of
K38A Dda under single cycle conditions. A, radiolabeled
30:15-mer DNA substrate was incubated with Dda in the presence or
absence of K38A Dda. The unwinding reaction was initiated by rapid
mixing with 5 mM ATP,10 mM
Mg(OAc)2, and 5 µM oligo(dT)15.
The oligo(dT)15 served to trap helicase that dissociated
from substrate during the reaction. Duplex substrate was separated from
ssDNA product by native 15% polyacrylamide gel electrophoresis, and
the amount of dsDNA and ssDNA in each sample was determined by using a
PhosphorImager. The data shown were obtained in the presence of 250 nM WT Dda and 30 nM K38A Dda. B,
unwinding of DNA (2 nM) by Dda (250 nM) in the
presence of 0 nM K38A Dda ( ), 30 nM K38A Dda
( ), 60 nM K38A Dda ( ), 125 nM K38A Dda
( ), 180 nM K38A Dda ( ), and 250 nM K38A
Dda ( ). The line through the data represents the best fit
to a single exponential.
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Dda and varying amounts of K38A Dda were incubated in reaction buffer
for 3 min, followed by incubation with substrate for an additional 5 min. The unwinding reaction was initiated upon addition of 5 mM ATP, 10 mM Mg(OAc)2, and 5 µM oligo(dT)15. Under single cycle
conditions, a change in the rate of unwinding would be expected if
hetero-oligomers were capable of unwinding the substrate at rates
different rates than the WT enzyme. The rate of unwinding of substrate
by 250 nM WT Dda in the presence of varying concentrations
of K38 Dda was similar to that in the presence of only WT Dda (Fig.
7A). The fact that no trend in
the rates for unwinding was observed suggests that hetero-oligomers do
not participate in the unwinding process. However, the amplitude for product formation is reduced with the addition of the mutant helicase (Fig. 6B). This result is expected if the mutant enzyme
competes for binding to the substrate, but the WT Dda that remains
productively bound is capable of unwinding the substrate in a single
cycle.

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Fig. 7.
Results from single cycle DNA unwinding by WT
Dda as a function of K38A mutant Dda. A, rates of
unwinding are plotted as a function of increasing concentration of K38A
Dda. Rates were obtained by fitting data in Fig. 6B to a
single exponential. B, the amplitude from each curve in Fig.
6B is plotted as a function of concentration of K38A Dda.
The dotted line above the data points represents
the ideal fit if Dda were acting as a monomer (n = 1),
whereas the dashed line below the data points is
the ideal fit for a dimeric helicase (n = 2), according
to Equation 1 (27). The line through the data is the best fit to
Equation 1, which provides a value for n of 1.3 ± 0.05.
|
|
The approximate reduction in the amplitude of product formation in the
presence of mutant Dda can be analyzed assuming a simple competition
for binding to substrate between WT Dda and K38A Dda. The amplitudes
were plotted as a function of the concentration of mutant Dda (Fig.
7B). For a simple competition, the quantity of productively
bound WT Dda can be calculated according to Equation 1 (27).
|
(Eq. 1)
|
A is the amplitude for unwinding by helicase under
single cycle conditions. AWT is the amplitude of
unwinding in the presence of WT Dda only (0.44, Fig. 6B).
[EWT] and [EMUT] are
the concentrations of WT and mutant helicase, respectively.
K
is the equilibrium binding
constant between WT Dda and 30:15-mer (31 nM, Fig.
4C). K
is the
equilibrium binding constants between K38A Dda and 30:15-mer (14 nM, Fig. 4C). If Dda functions as a monomer,
then the value for n will be 1, whereas if Dda functions as
an oligomer, then n will be 2 or greater (27). The best fit
of the data is shown, and provides a value for n of 1.3 ± 0.05 (Fig. 7B). For comparison, the dashed line below the data points in Fig. 7B shows the
expected reduction in amplitude if Dda functions as a dimer, whereas
the dotted line above the data points represents
the expected reduction in amplitude if Dda functions as a monomer.
Clearly, the data fall between these expected values. However, the
difference of only 2-fold between the expected results for a monomer or
dimer makes the observed data difficult to interpret for Dda. For
comparison, the rate and amplitude for unwinding was dramatically
reduced in an experiment reported for the NS3 helicase domain (27).
Unwinding by Dda in the Presence of K38A Dda under
Steady-state Conditions--
It is possible that more than one Dda
monomer can bind to the substrate under conditions of excess enzyme
reported in Fig. 6. In this case, the presence of K38A Dda might lead
to a reduction in unwinding due to steric interactions between Dda and
K38A Dda, rather than specific protein-protein interactions. To avoid
this possibility, unwinding reactions were performed in the presence of
excess substrate, in order to greatly reduce the likelihood that two
proteins would bind to the same substrate molecule, unless specific,
protein-protein interactions were involved.
Dda was incubated with 30:15-mer and the unwinding reaction was
initiated upon the addition of 5 mM ATP and 10 mM Mg(OAc)2. A 15-mer oligonucleotide that was
complementary to the displaced strand of the 30:15-mer substrate was
introduced along with the ATP to prevent reannealing of products.
Observed unwinding rates increased proportionally with 2.5, 5, and 7.5 nM Dda (Fig. 8A). Although the trapping strand is likely to sequester Dda, the observed rates of strand separation reflect the enzyme's activity. Therefore, if K38A Dda binds to WT Dda and lowers the effective activity of the WT
enzyme, then the observed rate for strand separation should reflect
this occurrence. Dda (5 nM) was incubated with varying
concentrations of K38A Dda, ranging from 0 to 40 nM for 2 min, followed by incubation with 30:15-mer (400 nM) for an
additional 5 min. The reaction was initiated upon addition of ATP and
Mg(OAc)2. The substrate was unwound with a rate of 1.42 nM·s
1 in the presence of 5 nM WT Dda (Fig. 8B). A comparison of unwinding rates at each concentration of K38A Dda is shown in Fig. 8C.
The rates of unwinding by 5 nM WT Dda in the presence of
10, 20, and 40 nM K38A Dda were 1.42, 1.68, and 1.66 nM·s
1, respectively. Thus, the
presence of K38A Dda at concentrations 8-fold higher than that of the
WT enzyme, did not decrease the observed rate of formation of ssDNA.
Additionally, 5 nM WT Dda alone or in the presence of 40 nM K38A Dda was incubated with 5 mM ATP and the
reaction was initiated by addition of 400 nM 30:15-mer.
Under these conditions WT Dda alone unwound the substrate at a rate of
1.35 nM·s
1, and WT Dda plus
K38A Dda unwound the substrate at a rate of 1.46 nM·s
1 (Fig. 8C).
Thus, no decrease in the rate of unwinding was observed under a variety
of conditions when competition for substrate was greatly reduced. These
results suggest that protein-protein interactions are not required for
unwinding to occur.

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Fig. 8.
Unwinding under steady state conditions in
the presence of varying concentrations of Dda or K38A Dda.
A, rate of strand separation of 30:15-mer substrate (400 nM) in the presence of 2.5, 5, and 7.5 nM Dda.
B, formation of product (ssDNA) by 5 nM Dda in
the presence of 0 nM K38A Dda ( ), 10 nM K38A
Dda ( ), 20 nM K38 Dda ( ), 40 nM K38 Dda
( ). Velocities were obtained by fitting the data to a linear
function (KaleidaGraph). C, plot of the rates for strand
separation obtained from panel B as a function of
varying concentrations of K38A Dda. Dda was incubated with varying
concentration of K38A Dda followed by incubation with 30:15-mer. The
reaction was inititated by addition of ATP ( ). Alternatively,
Dda was incubated with K38A Dda and ATP prior to initiation of the
reaction with 30:15-mer ( ).
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 |
DISCUSSION |
The search for a functional oligomeric structure of Dda has proven
elusive. Experimental approaches such as chemical cross-linking and gel
filtration did not strongly support nor eliminate formation of
oligomeric species (Figs. 1 and 2). Biochemical assays in which the
ATPase activity of Dda was measured at varying enzyme concentration did
not provide any evidence for oligomerization (Fig. 3). Hence, methods
that have provided support for oligomerization of other helicases gave
negative results with Dda. Transient, protein-protein interactions that
might be required for unwinding activity were not eliminated by these experiments.
To further investigate this, an ATPase-deficient mutant enzyme was
prepared that was unable to unwind DNA. If oligomerization were
required for function, then the mutant enzyme would be expected to
lower the activity of the wild type due to formation of
hetero-oligomers. The effects of an ATPase-deficient mutant Dda protein
(K38A Dda) on the activities of WT Dda were examined. The presence of
K38A Dda protein, which is inactive as a helicase, but is still capable of binding DNA (Fig. 4), fails to decrease the ATPase activity of WT
Dda (Fig. 5). The rate of unwinding under single cycle conditions was
not reduced by the addition of the mutant enzyme, indicating that
hetero-oligomers do not likely participate in the unwinding reaction.
The amplitude for unwinding by WT Dda helicase was reduced upon
addition of K38A Dda (Fig. 6). The data fall between that expected for
a monomeric or dimeric helicase when analyzed as though a simple
competition for substrate binding existed between wild type Dda and
mutant or hetero-oligomeric Dda (Fig. 7B). It is possible
that more than one Dda monomer can bind to the substrate without
invoking protein-protein interactions, which may lead to a greater than
expected reduction in amplitude under conditions of excess enzyme and
in the presence of the inactive K38A Dda.
The effect of mixing K38A Dda and WT Dda was further investigated by
conducting unwinding experiments in the presence of excess substrate.
Under these conditions, the competition for DNA binding between the two
proteins is effectively eliminated, because the protein will be
distributed among the substrate molecules. If Dda is acting as a
monomer, the addition of mutant protein should have no effect on WT Dda
unwinding activity under these conditions because the mutant protein
will bind to DNA, but will not prevent WT Dda binding and subsequent
unwinding activity. However, if protein-protein interactions are
required for unwinding, then such interactions should occur, despite
the presence of excess DNA substrate. The results indicate that the
presence of mutant Dda does not reduce the rate of unwinding (Fig.
8C), suggesting that Dda may not require oligomerization in
order to function. Even when WT Dda alone or in the presence of K38A
Dda is incubated with ATP prior to initiating the reaction with
substrate, there is no reduction in the unwinding rate (Fig.
8C). Thus, Dda appears to be capable of functioning as a
monomer. This may explain the fact that Dda is known to act in a
distributive manner, cycling on and off of DNA during unwinding of long
substrates (19). Perhaps this distributive mode of action is the result
of a monomeric structure that does not allow Dda to encircle ssDNA in a
manner similar to that of many hexameric helicases (4, 18, 22).
In order to unwind a region of dsDNA, and translocate to a new site
along the nucleic acid, helicases need two DNA binding sites so that
the enzyme does not dissociate during unwinding. Therefore, Dda is
expected to contain more than one site that is capable of binding to
DNA. An inchworm mechanism has been suggested for unwinding and
translocation by monomeric helicases. Evidence for multiple DNA binding
sites on a monomeric helicase has been provided in the case of the PcrA
helicase. The crystal structure of PcrA in the presence of a partial
duplex DNA substrate indicates a binding site for ssDNA and an adjacent
binding site for dsDNA (7). Dda is classified as a super family 1 helicase, like PcrA, based on sequence comparison (11), although Dda is
a 5'-to-3' helicase (17), whereas PcrA translocates 3'-to-5' (30).
Another possible arrangement for "two" binding sites has been
proposed for the NS3h helicase. The co-crystal structure of this enzyme
in the presence of ssDNA indicates that 7 nucleotides are complexed
within a groove between two adjacent domains and a third domain (31).
The two domains on one side of the groove were proposed to interact
with the ssDNA such that one domain could move relative to the other as
a function of ATP binding and hydrolysis. Hence, the ssDNA bound in one
domain could be released during translocation while interactions in the
second domain are maintained to hold onto the DNA. Whether one of the models suggested for PcrA or NS3h applies to Dda remains to be determined.
Helicases in superfamily 1 and superfamily 2 have been difficult to
characterize in terms of oligomeric structure, and much discussion has
surrounded this issue. This is due to the fact that many biophysical
approaches for studying oligomerization provide negative results when
applied to these enzymes. Evidence has been presented that suggests
that PcrA (7), UvrD (8), and NS3h (32) can function as monomers.
Another helicase, PriA, was shown to exist as a monomer in solution,
even when bound to ssDNA, although the functional form of the enzyme
has not been determined (33). Others have provided evidence that
suggests that oligomeric forms of UvrD (34) and NS3h (27) are required for optimal unwinding activity.
Previously, the ability of Dda to displace streptavidin from the 3' end
of biotin-labeled oligonucleotides was found to exhibit a dependence on
the length of the oligonucleotide. The streptavidin displacement
reaction was found to proceed faster from longer oligonucleotides than
from shorter ones (18). The results described here suggest that a
monomeric form of Dda can function to unwind DNA. However, individual
Dda monomers may align along the nucleic acid lattice to enhance
overall activity in the streptavidin displacement assays, despite the
lack of strong protein-protein interactions.