DnaB from Thermus aquaticus Unwinds Forked Duplex DNA
with an Asymmetric Tail Length Dependence*
Daniel L.
Kaplan
§ and
Thomas A.
Steitz
¶
**
From the Departments of
Molecular Biophysics and
Biochemistry and ¶ Chemistry, Yale University and the
Howard Hughes Medical Institute,
New Haven, Connecticut 06520-8114
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ABSTRACT |
DnaB helicase is a ring-shaped hexamer of 300 kDa
that is essential for replication of the bacterial chromosome. The
dnaB gene from Thermus aquaticus was isolated
and cloned, and its gene product was expressed and purified to
homogeneity. A helicase assay was developed, and optimal conditions for
T. aquaticus DnaB activity were determined using a forked
duplex DNA substrate. The activity required a hydrolyzable nucleoside
triphosphate and both 5'- and 3'-single-stranded DNA tail regions.
Under conditions of single enzymatic turnover, the lengths of the 5'-
and 3'-single-stranded regions were varied, and 6-10 nucleotides of
the 5'-single-stranded tail and 21-30 nucleotides of the
3'-single-stranded tail markedly stimulated the unwinding rate. These
data suggest that DnaB from T. aquaticus interacts with
both DNA single-stranded tails during unwinding and that a greater
portion of the 3'-tail is in contact with the protein. Two models are
consistent with these data. In one model, the 5'-single stranded region
passes through the central hole of the DnaB ring, and the 3'-tail makes
extensive contact with the outside of the protein. In the other model,
the 3'-single-stranded region passes through the DnaB ring, and the
outside of the protein contacts the 5'-tail.
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INTRODUCTION |
DnaB, a ring-shaped hexamer of ~300 kDa (1-3) is essential for
replication of the bacterial chromosome (4). It is also involved in the
replication of bacterial phage (5, 6) and plasmid DNA (7). It has been
proposed that the protein unwinds double-stranded DNA at a replication
fork, providing single-stranded DNA templates for DnaG primase and DNA
polymerase III holoenzyme (8). The protein exhibits helicase activity
in vitro, catalyzing the conversion of double-stranded DNA
to single-stranded DNA with concomitant hydrolysis of ATP (9, 10).
DnaB is homologous to two other characterized hexameric helicases
involved in DNA replication, the gene 4 protein from T7 phage and the
gene 41 protein from T4 phage (11, 12). It has been shown that members
of this helicase family unwind DNA with 5' to 3' polarity and require
both 5'- and 3'-single-stranded tail regions for efficient unwinding of
duplex DNA (10, 13-17). These proteins are highly processive and may
stay bound to the DNA throughout replication (18, 19). Electron
microscopy studies of Escherichia coli DnaB and gene 4 from
T7 have demonstrated that these protein hexamers are ring-shaped with a
central hole that is wide enough to fit single-stranded DNA (2, 3, 20, 21). In the case of the T7 gene 4 protein, it has also been shown by
electron microscopy that a DNA strand is present in the central hole of
the protein ring (20, 21).
We are interested in studying the mechanism of DnaB-catalyzed unwinding
by determining the crystal structure of DnaB in complex with forked
duplex DNA. Proteins from thermophilic organisms are often easier to
purify and may crystallize more readily than their mesophilic
counterparts. Therefore, we isolated and cloned the gene for
Thermus aquaticus dnaB, overexpressed its gene product, and
purified the protein to homogeneity.
To design DNA substrates for cocrystallization with DnaB and to begin
establishing its mechanism, we need to know what single-stranded regions of DNA are contacting the protein during unwinding. To address
this issue, we developed a helicase assay using a forked duplex DNA
substrate. We then varied the lengths of the single-stranded tail
regions and found that the first 6-10 nucleotides of the 5'-tail and
the first 21-30 nucleotides of the 3'-tail emanating from the duplex
region markedly stimulated the unwinding rate. Thus, these data suggest
that the 3'-tail makes more extensive contacts with T. aquaticus DnaB than the 5'-tail. Two models may account for these
data. In one, the 5'-single-stranded tail passes through the central
hole of the protein hexamer, whereas the 3'-tail makes more extensive
contact with the outside of the protein toroid. In the second model,
the 3'-tail passes through the central hole, and the 5'-tail makes less
extensive contact with the outside of the protein ring.
These two alternative models would position the protein ring around the
lagging or leading strands of the DNA, respectively, during replication.
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EXPERIMENTAL PROCEDURES |
Isolation and Cloning of the dnaB Gene from T. aquaticus
Genomic DNA from T. aquaticus YT-1 strain was
prepared as described previously (22). To obtain the initial
PCR1 product, 50-µl
reactions were set up with 50 ng of genomic DNA, 20 µM
each primer, 3.3 mM dGTP and dCTP, 1.7 mM dATP
and dTTP, 1× Vent buffer (New England Biolabs), and 1 µl of Deep
Vent DNA polymerase (New England Biolabs). The forward primer was
GG(GC)TC(GC)ATCGAGCAGGACGC(GC)GA(CT), which was designed to include the
amino acid region corresponding to 406 to 413 of DnaB from E. coli. The reverse primer was 5'-(GC)GG(GC)CCGTT(GC)CGCTG(CT)TT-3', which was designed to be complementary to residues 439-445. For the
PCR, the melting conditions were 1 min at 95 °C, the annealing conditions were 1 min at a temperature that was decreased stepwise from
60 to 45 °C in 0.5 °C increments (31 cycles), and the extension conditions were 2.5 min at 72 °C.
To isolate regions 5' and 3' to the initial product, inverse PCR was
performed. Templates for inverse PCR were prepared by digesting
T. aquaticus genomic DNA with various restriction enzymes. The restriction enzymes were chosen to yield products of average size
between 500 and 2000 base pairs. The average size product was estimated
by determining the frequency of a particular restriction site within
all T. aquaticus genomic DNA sequence that had been deposited in the data base. After the restriction enzyme had been heat
inactivated, the digest was religated at a low (2 nM) DNA concentration to favor unimolecular reactions, thus yielding circular fragments of genomic DNA. Ligation reactions were performed with T4 DNA
ligase (New England Biolabs) according to the instructions of the
manufacturer. Inverse PCR was performed using primers directed away
from each other and against regions of dnaB that had been sequenced. The PCRs were set up as above except that the annealing temperature was decreased in 0.3 °C increments from 70 °C to
60 °C (35 cycles). Pfu DNA polymerase (Stratagene)
instead of Deep Vent DNA Polymerase was used in some of these inverse PCRs.
To isolate the entire gene, rTth DNA polymerase, XL (Perkin-Elmer) was
used. The forward primer contained the N terminus of the gene, and the
reverse primer was complementary to a region 3' to the C terminus. The
PCR was performed as above with a 5-min extension time. The full-length
gene was cloned into a pET-22b(+) expression vector (Novagen) using
NdeI and SacI restriction sites.
Expression and Purification of the dnaB Gene Product from T. aquaticus
Expression, Lysis, and Heat Treatment--
The pET22b(+) vector
containing the dnaB gene was transformed into BL-21 cells.
The transformed cells were grown in 10 liters of LB medium containing
50 µg/ml ampicillin in a stirred, aerated fermentor at 37 °C. When
the cells reached an A600 of 0.6, the temperature was decreased to 30 °C, and
isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 100 µM to induce gene expression. Three
hours later, 34 g of cells were harvested by centrifugation. From
this point, all manipulations were carried out at 4 °C unless otherwise stated. The 34 g of cells were resuspended in 200 ml of
Buffer A, which contained 10% sucrose, 50 mM Tris-HCl, pH
7.5, 50 mM NaCl, and 5 mM DTT. The cells were
then lysed with a French press. MgCl2 was then added to a
final concentration of 5 mM. The lysate was then heated at
65 °C for 20 min followed by chilling on ice for 20 min. The cells
were then spun at 38,000 rpm in a Ti45 rotor (Beckman) for 60 min. The
supernatant was filtered with a 0.22 µm low protein binding filter
(Fraction I).
Q Source Column--
A Q source column (Amersham Pharmacia
Biotech) was preequilibrated with Buffer B, which contained 10%
glycerol, 50 mM Hepes, pH 7.0, 50 mM NaCl, 5 mM MgCl2, and 5 mM DTT. Fraction I
was loaded onto the column and the column was then washed with 5 column
volumes of Buffer B. The protein was eluted with a linear gradient of Buffer B to Buffer B containing an additional 450 mM NaCl.
DnaB eluted at 300-345 mM NaCl (Fraction II).
Heparin-Sepharose Column--
Fraction II was dialyzed against
Buffer C, which contained 10% glycerol, 20 mM Hepes, pH
7.0, 50 mM NaCl, 5 mM MgCl2, 5 mM DTT, and 0.02% sodium azide. After dialysis, the sample
was loaded onto a HiTrap heparin-Sepharose column (Amersham Pharmacia
Biotech) that was preequilibrated with Buffer C. The column was then
washed with 5 column volumes of Buffer C followed by elution with a
linear gradient of Buffer C to Buffer C containing an additional 450 mM NaCl. DnaB eluted between 200 and 250 mM
NaCl (Fraction III).
Concentration--
Fraction III was dialyzed against Buffer D,
which contained 10% glycerol, 20 mM Hepes, pH 7.0, 100 mM NaCl, 5 mM MgCl2, 5 mM DTT, and 0.02% sodium azide. The sample was then
concentrated with an Amicon Ultrafiltration device followed by an
Amicon Centricon device. The concentration of protein was 11.1 mg/ml at
this point. The final yield was 112 mg of purified protein from a
10-liter cell growth. 80% glycerol was added to the sample to achieve
a final concentration of 50% glycerol. The sample was then stored at
20 °C until further use.
Protein Analysis
A reducing, 10-20% polyacrylamide gel was run in SDS using the
Laemmli buffer system (23). The gel was run at a constant 175 V for 45 min and then stained with Coomassie Blue as described (24). Molecular
weight marker standards were from Amersham Pharmacia Biotech (catalog
no. 17-0446-01).
Accurate protein concentration and amino acid content were determined
by performing amino acid analysis on triplicate samples of purified
protein. This analysis was carried out by the Howard Hughes Medical
Institute Biopolymer/W. M. Keck Biotechnology Resource Laboratory
at Yale University (Keck Facility) on a Beckman model 6300 ion exchange
instrument following acid hydrolysis.
DNA Analysis
All DNA sequencing was performed by the Keck Facility using
Applied Biosystems 377 DNA sequencers. The sequence reactions utilize
fluorescently labeled dideoxynucleotides (dRhodamine Terminators) and
Taq FS DNA polymerase in a thermal cycling protocol.
Helicase Assay
Substrate Preparation--
All DNA oligomers were synthesized by
the Keck Facility and gel-purified. The 3'-tailed oligomer was labeled
at the 5'-end with T4 polynucleotide kinase (New England Biolabs). The
kinase was denatured by heating to 65 °C for 20 min. The
unincorporated nucleotides were removed with a G-50 Sephadex spin
column. This labeled strand was mixed at a 1:2 ratio with the
unlabeled, complementary strand. Identical helicase reaction rates were
obtained when this ratio was 1:1 (data not shown), and the excess
unlabeled strand was added to enhance annealing. Two equivalent
unlabeled strands of the same 1:2 ratio were added to the labeled
mixture in a 5-fold excess. The unlabeled mixture was added in excess
to ensure that the concentration of substrate was accurate. The strands
were then annealed by heating to 95 °C for 5 min followed by slow
cooling to room temperature. The strands were then preequilibrated at 55 °C for 3 h. This step ensured that the percentage of duplex remained constant throughout the assay in the absence of enzyme.
Assay Conditions--
Standard conditions for the helicase assay
were as follows: 500 nM T. aquaticus DnaB
helicase, 5 mM GTP, 1 nM DNA substrate, 10%
glycerol, 25 mM sodium Bicine, pH 9.0, 50 mM
potassium glutamate, 10 mM NaCl, 10 mM
MgCl2, 1.5 mM DTT, 0.1 mg/ml bovine serum
albumin, and 55 °C. The reagents in the assay were added in the
following order on ice: buffer, DNA substrate, enzyme, and finally GTP. After incubation at 55 °C for the time indicated in the text, the
reactions were quenched by placing on ice and adding stop buffer to
achieve a final concentration of 1% SDS, 40 mM EDTA, 20%
glycerol, and 0.1% xylene cyanol. The samples were then snap frozen in
liquid nitrogen and stored at
20 °C until they were ready for
final analysis by native polyacrylamide gel electrophoresis.
Single strand product was separated from duplex substrate by running
the samples through a 12% polyacrylamide gel (19:1 acrylamide: bis) in
1× TBE (90 mM Tris-Borate, 2 mM EDTA) at 5 W
at room temperature for 1-2 h. The variation in time depended upon the
lengths of the DNA species in the assay.
Quantitation of Product--
After the gel was run, it was dried
at 50 °C for 40 min. The gel was then exposed to a Bio-Imaging Plate
(Fuji Photo Film Co.). Band intensities were quantified and background
counts subtracted using the MacBAS software package. The percentage of
single strand was typically ~ 0-10% in an unreacted sample and
~95-100% in a heat-denatured sample. To normalize for the slight
variability in these values, the percentage of product was calculated
using the following equation,
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(Eq. 1)
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where %SSs is the percentage of single strand in
the sample lane of interest, %SSd is the percentage of
single strand in the unreacted duplex lane, and %SSh is
the percentage of single strand in the heat-denatured lane.
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RESULTS |
Isolation, Cloning, Expression, and Purification of DnaB from T. aquaticus--
The dnaB gene from T. aquaticus
isolated and cloned using the technique of PCR. The complete amino acid
sequences of DnaB from five other bacterial species had been deposited
in the data base at the time of cloning. Based on these sequences and
the codon usage frequency of T. aquaticus, degenerate
oligonucleotide primers for PCR were designed (25, 26). Using genomic
DNA as a template, a 120-base pair DNA fragment was initially isolated and sequenced. This fragment encoded an amino acid sequence that it is
highly similar to DnaB from other bacterial species within the region
between the primers. The technique of inverse PCR was then used to
isolate regions 5' and 3' to the original sequence (27). The N terminus
of the gene was identified by a Shine-Dalgarno sequence (GGTAGG)
situated 10 base pairs upstream to an ATG initiation codon. This
5'-region was amplified and sequenced in triplicate because it was used
as a primer for subsequent PCR gene amplification. The entire gene was
PCR-amplified in triplicate using one primer containing the
5'-initiation site and a second primer complementary to a region 3' to
the C terminus. These three products were then cloned and sequenced.
Because amplification and sequencing were performed in independent,
triplicate experiments the gene sequence was unambiguously confirmed.
The predicted amino acid sequence of the dnaB gene product
yields a protein of 49 kDa per monomer. The GC content of the gene is
67%, typical of genes from this species. The protein has 48% amino
acid identity and 69% amino acid similarity compared with DnaB from
E coli. Regions within this gene that are highly conserved among other species (12) are well conserved for T. aquaticus DnaB. The protein has 1 cysteine and 14 methionines, which may be
useful in future crystallography work.
The T. aquaticus dnaB gene was then cloned into a T7
expression system (28). Induction and purification of the DnaB gene product were carried out as described under "Experimental
Procedures." The final protein sample was ~ 95% pure as
judged by Coomassie Blue staining of a reducing SDS-polyacrylamide gel
electrophoresis gel (not shown). The protein ran according to its
predicted molecular weight on this gel. Once purified product was
obtained, amino acid analysis was performed to confirm the correct
identity of the product and to determine an accurate extinction
coefficient, which was calculated to be
280 = 0.46 liter
g
1 cm
1.
Development of a Helicase Assay--
To determine what 5'- and
3'-tail lengths of a forked duplex DNA substrate are bound by T. aquaticus DnaB during unwinding, we developed a helicase assay. In
this assay, the rates of unwinding under conditions of single enzyme
turnover are measured for a series of forked duplex DNA substrates with
various 5'- and 3'-single-stranded tail lengths. As the tail is
shortened beyond a length that is critical for binding and/or
unwinding, a marked decrease in helicase rate should be observed.
The standard substrate used in this helicase reaction had a 22-base
pair duplex region and contained a 30-nucleotide deoxythymidylate (d(pT)30) in each single-stranded tail region (Fig.
1). The duplex region had a melting
temperature of ~65 °C, and the poly(T) base composition of either
tail ensured that there was no interaction between the tails. The
strand with the 3'-tail was labeled at the 5'-end with 32P,
and the conversion of this strand from duplex to single strand was
monitored with native gel electrophoresis.

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Fig. 1.
Standard substrate used in the helicase
assay. This DNA substrate has a 22-base pair duplex region, a
30-nucleotide deoxythymidylate 5'-single-stranded tail region
(d(pT)30-5'), and a 30-nucleotide deoxythymidylate
3'-single-stranded tail region (d(pT)30-3'). *, position of
the 32P labeling.
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Conditions for the helicase assay were optimized to achieve a maximum
rate of product formation. These conditions are described under
"Experimental Procedures." Briefly, 500 nM enzyme, 1 nM DNA substrate, and 5 mM GTP were incubated
at pH 9.0 at 55 °C for various times. A low substrate concentration
was used to slow the rate of product reannealing. Furthermore, enzyme
concentration was in vast excess compared with substrate concentration,
ensuring that the kinetics observed were for single-enzyme turnover.
Multiple-turnover kinetic data can be dominated by
Koff rates and may be inadequate to assess
productive binding. A time course for this helicase assay was then
performed (Fig. 2A, lanes
1-13, and Fig. 2B). Nearly 50% of the substrate was
converted to product in 2 min in the presence of 5 mM GTP,
but there was no observable unwinding without added GTP.

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Fig. 2.
Time course of the helicase reaction.
A, native gel analysis of T. aquaticus DnaB
unwinding the standard substrate shown in Fig. 1. Lanes 1 and 2 contain the double-stranded substrate and the
3'-tailed single-stranded product, respectively. Lane 3 contains double-stranded substrate that was heated to 95 °C for 5 min. Lanes 4-8 and 9-13 contain reactions that
were performed in the absence and presence of 5 mM GTP,
respectively. Lane 14 contains the 3'-tailed single-stranded
product annealed to a 22-nucleotide DNA trap oligomer that is
complementary to the duplex region but contains no 5'-single-stranded
tail region. Lanes 15-19 contain reactions that were
performed in the absence of DnaB and in the presence of 40 nM of this trap DNA oligomer. B, the bands in
A were quantified and the percentage of product calculated
according to Equation 1 under "Experimental Procedures." This graph
depicts the first 30 min of unwinding in the presence of GTP (A,
lanes 9-12). C, lanes 15-19 of
A were quantified, and the change in percentage of substrate
as a function of time is shown.
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Although protein-dependent conversion of double-stranded
DNA to single-stranded DNA is the hallmark of helicase activity, if the
substrate and product are in rapid exchange at equilibrium, a reagent
with single-stranded binding activity can sequester the product and
drive the equilibrium toward product formation. This nonenzymatic
activity is seen for single-stranded binding proteins such as E. coli single-stranded binding protein and T4 gp32 (29). To
determine whether this could occur under these assay conditions, a
22-nucleotide single-stranded DNA oligomer that was complementary to
the duplex region of the labeled strand was added to the reaction in
20-fold excess. This "trap" DNA oligomer contained no
5'-single-stranded tail. If melting occurs, this excess DNA should bind
to the released labeled single strand and inhibit the reverse reaction.
Because this excess unlabeled strand contained no tail region, its
duplex with labeled strand could be electrophoretically separated from
substrate. After 2 h, there was only a ~6% decrease in
substrate concentration, suggesting that the substrate and product were
not in rapid exchange relative to the time course of the reaction (Fig.
2A, lanes 14-19, and Fig. 2C).
Reaction Requirements for Helicase Activity--
Enzymatic
unwinding is characterized by coupled hydrolysis of a nucleoside
triphosphate. To determine whether the helicase activity mediated by
T. aquaticus DnaB was dependent upon nucleoside triphosphate
hydrolysis, the reaction was performed in the presence of protein and
various nucleotide analogs (Fig.
3A). After a 30-min incubation, nearly all of the substrate was converted to product in the
presence of 5 mM ATP or GTP. By contrast, incubation with the same concentration of the nucleoside diphosphate GDP yielded no
product. Similarly, incubation with the slowly hydrolyzable analogs
GTP
S, AMP-PNP, and AMP-PCP yielded only 9, 4, and 3% product,
respectively, after 30 min. These data suggest that hydrolysis of a
nucleoside triphosphate is required for helicase activity by T. aquaticus DnaB. Incubation with either ADP or ATP
S resulted in
accumulation of radiolabel in the well of the gel ("well shift") that was difficult to reverse. We therefore cannot assess the enzymatic
conversion to product in the presence of these analogs.

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Fig. 3.
Nucleotide requirement for the helicase
reaction. The standard substrate shown in Fig. 1 was used in these
30-min reactions. A, native gel analysis of the helicase
reaction performed in the presence of 5 mM of various
nucleotides and nucleotide analogs. B, the helicase reaction
was performed in the presence of 5 mM of various nucleoside
triphosphates. Analyses were performed as described under
"Experimental Procedures."
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Other nucleoside triphosphates could stimulate the helicase activity of
DnaB. As shown in Fig. 3B, any of the ribonucleoside triphosphates resulted in nearly complete conversion to product in 30 min, whereas the deoxyribonucleoside triphosphates were generally less effective.
The protein concentration dependence for the reaction, expressed in
hexamers, is shown in Fig. 4A.
DnaB at a concentration of 5 nM resulted in nearly 50%
product in 30 min, and 50 nM DnaB resulted in nearly 90%
conversion to product. These concentrations were in excess of
substrate, which was at 1 nM concentration. The large
excess of protein required here for activity is atypical for an
enzymatic reaction. However, this relationship is typical for the
hexameric helicases (10, 13, 16). We also noted a log/linear
relationship between protein concentration and activity. Again, this is
not typical of most enzymatic reactions, but it has been reported for
the T4 gene 41 helicase (13). The unusual concentration dependence for
hexameric helicase activity may be explained if the enzyme dissociates
at low protein concentrations.

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Fig. 4.
Reagent requirements for the helicase
reaction. The standard substrate shown in Fig. 1 was used in these
reactions. A, the percentage of product as a function of
hexamer concentration is shown for 30-min incubations. B,
the percentage of product as a function of pH is shown for 30-min
incubations. The buffers used were sodium acetate (5.0), sodium Mes
(6.0), sodium Hepes (7.0 and 8.0), or sodium Bicine (9.0).
C, the percentage of product as a function of potassium
glutamate concentration is shown for 8-min (open circles)
and 30-min (filled circles) incubations.
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There was a steep pH dependence for activity, with the protein was far
more active under alkaline conditions (Fig. 4B). Greater than 90% conversion to product was observed at pH 8 or 9. Because the
activity increase was largest in the pH range 5-8, it is more likely
that some group(s) on the protein was being titrated as opposed to the
DNA because DNA has no group with a pKa in this pH range.
The salt dependence of the reaction rate was also determined. The
addition of 50 mM potassium glutamate resulted in a slight enhancement of activity compared with 0 mM potassium
glutamate (Fig. 4C). However, each reaction contained 10 mM NaCl from the protein storage buffer, as well as 25 mM sodium Bicine and 10 mM MgCl2.
Higher concentrations of potassium glutamate were inhibitory in a
concentration-dependent manner (Fig. 4C). These
data are consistent with salt-mediated disruption of a critical ionic
interaction, possibly between the protein and the DNA. Replacing
potassium glutamate with sodium glutamate, sodium chloride, or
potassium chloride resulted in very little change in the percentage of
product formed (not shown).
Helicase Rates Using Single-tailed Substrates and Estimation of
Reannealing Rates--
To examine the role of the 5'- and
3'-single-stranded tail regions in the reaction, the assay was
performed with a substrate containing a d(pT)30-3'-tail and
no 5'-tail or with a substrate containing a d(pT)30-5'-tail
and no 3'-tail. In either case, there was no conversion to product
within a 2-h time course, indicating that both tails are required for
activity (not shown). These data further support the claim that DnaB is
acting as a helicase and not a single-stranded binding protein because
it is unlikely that product binding is
two-tail-dependent.
A helicase assay is complicated by the fact that the single-stranded
products can reanneal to form substrate. Thus, it is important to
measure the rate of product reannealing. A substrate containing a
d(pT)30-5'-tail and no 3'-tail was heat-denatured and then
allowed to reanneal in the absence and presence of protein under the
conditions used in these assays. This substrate was chosen because DnaB
could not unwind it. Without protein, the half-time for reannealing was
~30 min (Fig. 5). However, the
half-time time for reannealing increased to ~ 120 min in the
presence of DnaB, suggesting that the enzyme inhibits reannealing. This
inhibition may be caused by transient protein interaction with the
single-stranded products (30, 31). Because the results presented here
are based upon the first 30 min of the reaction, product reannealing will not substantially alter the reaction kinetics presented. However,
the percentage of product reported will slightly underestimate the
total product produced, particularly at 30-min time points.

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Fig. 5.
Estimation of single-stranded product
reannealing rates. A substrate identical to that shown in Fig. 1
except with no 3'-single-stranded tail region was heated to 95 °C
for 5 min. The single-stranded products were then allowed to reanneal
under the standard experimental reaction conditions in the absence
(open circles) or presence (filled circles) of
500 nM T. aquaticus DnaB.
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Effect of 5'- and 3'-Tail Length on Helicase Rate--
The length
of the 5'-tail was then varied stepwise from d(pT)30 to
d(pT)0, whereas the 3'-tail length remained constant at d(pT)30. There was a very slight decrease in helicase rate
as the length of the 5'-tail was decreased stepwise from
d(pT)30 to d(pT)10 (Fig.
6A). However, there was a
substantial decrease as the 5'-tail length was shortened from
d(pT)10 to d(pT)5 and from d(pT)5
to d(pT)2. Thus, from the duplex region, the first 6-10
deoxythymidylates of the 5'-tail markedly stimulate unwinding, but
d(pT)s 11-30 have little effect. These data suggest that only the
first 6-10 d(pT)s of the 5'-tail productively interact with DnaB
during this helicase reaction.

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Fig. 6.
Effect of 5'- or 3'-tail length on the
helicase rate. A and B, the length of the
5'-single-stranded tail region (A) or the 3'-single-stranded
tail region (B) of the standard substrate shown in Fig. 1
was decreased in stepwise increments (d(pT)30, filled
circles; d(pT)20, open circles;
d(pT)15, filled squares; d(pT)10,
open squares; d(pT)5, filled
triangles; d(pT)2, open triangles). The
other single-stranded tail region was a constant d(pT)30.
C, helicase rates for substrates containing
d(pT)30-5' and d(pT)30-3'-tails (filled
circles), d(pT)10-5' and d(pT)30-3'-tails
(filled triangles), d(pT)30-5' and
d(pT)10-3'-tails (open triangles), or
d(pT)10-5' and d(pT)10-3'-tails (×).
D, native gel analysis of the helicase reaction
performed using a substrate containing d(pT)10-5' and
d(pT)30-3'-tails (lanes 1-10, 10-T-5'_30-T-3'),
or d(pT)30-5' and d(pT)10-3'-tails (lanes
11-20, 30-T-5'_10-T-3').
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The length of the 3'-tail was then varied stepwise from
d(pT)30 to d(pT)0 while the 5'-tail length
remained constant at d(pT)30. There was a measurable
decrease in helicase rate as the 3'-tail length decreased stepwise from
d(pT)30 to d(pT)0, suggesting that most of this
region stimulated the unwinding rate and therefore may contact the
protein during the reaction (Fig. 6B).
A substrate with a d(pT)10-5'-tail and a
d(pT)10-3'-tail was converted to product at the same rate
as one containing a d(pT)30-5'-tail and a
d(pT)10-3'-tail (Fig. 6C). These data suggest
that the stimulatory effect of d(pT)s 11-30 of the 3'-tail is
independent of d(pT)s 11-30 of the 5'-tail. Likewise, the inability of
d(pT)s 11-30 of the 5'-tail to stimulate the unwinding rate is
independent of d(pT)s 11-30 of the 3'-tail.
The above results suggest that DnaB from T. aquaticus
contacts the first 6-10 nucleotides of the 5'-tail and the first
21-30 nucleotides of the 3'-tail as the helicase unwinds duplex DNA. Thus, DnaB may have more extensive contact with the 3'-tail than the
5'-tail during unwinding. This possibility is best exhibited by the
substantially increased unwinding rate of the forked duplex containing
a d(pT)10-5'-tail and a d(pT)30-3'-tail
compared with one containing a d(pT)30-5'-tail and a
d(pT)10-3'-tail (Fig. 6D).
The unwinding of these substrates was also compared in reactions
containing ATP instead of GTP because ATP is likely to be the
predominant cellular cofactor. When these samples were analyzed by
native gel electrophoresis, a substantial well shift was seen under
several conditions (Fig. 7A).
Therefore, all of the samples were incubated with 20 mM of
the single-stranded oligomer d(pT)23 for 1 h at room
temperature and reanalyzed. Incubation with this oligomer effectively
removed the well shift but did not alter the single strand percentage
in lanes with no well shift (Fig. 7B). Again, nucleotides
11-30 of the 3'-tail markedly stimulated the unwinding rate, but
nucleotides 11-30 of the 5'-tail had little effect. Thus, a similar
asymmetric tail length dependence was found for reactions containing
either GTP or ATP as a cofactor (Fig. 7, B and
C).

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Fig. 7.
Helicase rates using ATP as a cofactor.
The reactions were performed as described under "Experimental
Procedures" except that 5 mM ATP replaced 5 mM GTP. A and B, native gel analyses
of reactions performed using substrates identical to the standard one
shown in Fig. 1, except that the 5'-tail was shortened by 20 nucleotides (lanes 1-10) or the 3'-tail was shortened by 20 nucleotides (lanes 11-20). In B, the reaction
samples were incubated with 20 mM of d(pT)23
single-stranded DNA for 1 h at room temperature before gel
loading. C, reaction rates using substrates containing a
d(pT)30-5'-tail and d(pT)30-3'-tail
(filled circles), a d(pT)10-5'-tail and
d(pT)30-3'-tail (filled triangles, from B,
lanes 4-7), or a d(pT)30-5'-tail and
d(pT)10-3'-tail (open triangles, from B,
lanes 14-17).
|
|
Finally, the deoxythymidylates in either tail were replaced with
deoxyadenylates to determine whether there was a difference between
pyrimidine- and purine-containing tails. A similar asymmetric tail
length dependence was observed (Fig. 8).
Thus, DnaB from T. aquaticus may productively interact with
a greater region of the 3'-single-stranded tail region as compared with
the 5'-tail region. This effect is relatively insensitive to the
nucleoside triphosphate or the single-stranded tail sequence present in
the reaction.

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Fig. 8.
Helicase rates using
deoxyadenylate-containing single-stranded tails. The reactions
were performed as described under "Experimental Procedures" with 5 mM GTP as a cofactor. The substrates were identical to
those used in Fig. 7C except that deoxyadenylate replaced
deoxythymidylate in the tail regions.
|
|
 |
DISCUSSION |
Novel Tail Length Dependence Observed for DnaB Helicase
Activity--
The dnaB gene from T. aquaticus
was isolated and cloned, and its protein product was overexpressed and
purified to homogeneity. The gene product shares 48% identity and 69%
similarity compared with its E. coli homologue. A helicase
assay for this protein was developed using a forked duplex DNA
substrate. The single-stranded tail length dependence for unwinding was
determined under conditions of single-enzyme turnover to determine the
DNA regions that productively interact with DnaB helicase. These data
will be useful in designing substrates for cocrystallization studies.
From the duplex region, the first 21-30 nucleotides of the
3'-single-stranded tail and the first 6-10 nucleotides of the
5'-single-stranded tail substantially stimulate the unwinding rate of
T. aquaticus DnaB. Nucleotides 11-30 of the 5'-tail exhibit
no effect or slightly stimulate activity, depending upon the base
composition of the tails and the nucleotide cofactor used in the
experiment. Thus, a larger portion of the 3'-tail stimulates helicase
activity compared with the 5'-tail, a result that has not previously
been observed for proteins of this family.
Polarity of DnaB Family Helicases--
Helicases can exhibit
either 5' to 3' or 3' to 5' polarity with respect to the DNA strand
that they are bound to. Gene 41 protein from T4 phage and gene 4 protein from T7 phage have been shown to exhibit 5' to 3' polarity (13,
14, 16). There is both direct (10) and inferential (32, 33) evidence
that E. coli DnaB unwinds DNA with 5' to 3' polarity. Most
helicases require just one single-stranded tail region for duplex
unwinding. However, for members of the DnaB protein family, both
single-stranded tail regions are required (10, 14, 16). Thus, polarity
is more difficult to assess for these proteins because the protein is
contacting both tails, and under some conditions, E. coli
DnaB has been shown to exhibit 3' to 5' polarity (10). Furthermore, because the DNA tail regions used in these polarity experiments are
quite long, the specific DNA regions that interact with the helicase
cannot be adequately addressed with these studies alone.
Helicase Assay Data Are Complementary to Dissociation Constant and
Electron Microscopy Data--
Equilibrium dissociation constant data
for E. coli DnaB bound to DNA have been previously obtained
and are complementary to those of our unwinding assay. Fluorescence
data show that the site size for E. coli DnaB binding to
single-stranded DNA is 20 ± 3 nucleotides (34). Fluorescence
experiments have also been performed to determine how E. coli DnaB binds forked duplex DNA in the presence of the
nonhydrolyzable ATP analog AMP-PNP (35, 36). These studies conclude
that DnaB binds to either the 5'- or 3'-single-stranded tail regions,
but the protein does not bind to both tails simultaneously (36). There
was a 6-20-fold lower Kd for 5'-tail binding
compared with 3'-tail binding (35, 36). It was also concluded that DnaB
was in two opposite orientations with respect to the duplex region of
the DNA depending on the single-stranded tail to which the protein was
bound (36). It is difficult to directly relate these binding constant
data to those of our helicase assay for several reasons. First, the two-tailed dependence for DnaB unwinding observed here and elsewhere (10) was not seen in these steady-state binding experiments. Second, it
is not known with certainty which protein orientation is directed
toward unwinding the duplex region in these binding assays. Finally
AMP-PNP may render DnaB in a different conformational state compared
with ATP or GTP.
Electron microscopy studies have been performed with E. coli
DnaB and with gene 4 from T7. For both proteins, the 3-dimensional shape of the hexamer is that of a ring-shaped structure with an internal cavity diameter of ~ 25-40 Å, which is large enough
to accommodate single-stranded DNA (2, 3, 20, 21). These proteins are
highly processive, and they may remain bound to the DNA throughout
replication (18, 19). High processivity may be conferred by having one
of the DNA single strands pass through the hexamer central cavity. In
the case of the T7 gene 4 protein, single-stranded DNA is passing
through the central hole of the protein ring in the electron microscopy
image (20, 21). Recently, fluorescence data have indicated that a DNA
single strand passes through the inner channel of E. coli
DnaB (37).
Two Possible Models for DnaB Interaction with a Replication
Fork--
Two likely models describe T. aquaticus DnaB
interaction with a replication fork. In the first model, the 5'-single
stranded tail region passes through the central hole of the protein
hexamer, whereas the 3'-tail contacts the outside of the protein (Fig. 9A). This model positions the
protein around the lagging strand during DNA replication. From the
replication fork, only the first 6-10 nucleotides of the strand that
passes through the interior of the protein would productively interact
with the helicase during unwinding. Moreover, the 21-30 nucleotides of
the strand that does not pass through the protein, the leading strand,
may contact the outside of the protein. This model is consistent with
the 5' to 3' polarity that has been demonstrated for E. coli
DnaB and other members of this helicase family. However, the length of
the 5'-single-stranded tail region of DNA that productively interacts
with the interior of DnaB in this model, 6-10 nucleotides, is somewhat
less than the single-stranded DNA length bound by E. coli
DnaB, 20 ± 3 nucleotides (34). It is possible that the region of
5'-single-stranded tail DNA located ~11-20 nucleotides from the
duplex passes through the central cavity of DnaB but does not
productively contact the protein as measured by this unwinding
assay.

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Fig. 9.
Two schematic models of DnaB
unwinding a replication fork. In both models, DnaG primase is
shown as a circle, and it contacts both DnaB and the lagging
strand. A, DnaB is represented as a cylinder
encircling the lagging strand of the replication fork. Relative to the
fork opening, the protein contacts the first 6-10 nucleotides of the
strand that passes through its interior and the first 21-30
nucleotides of the strand that does not pass through its center.
B, DnaB is encircling the leading strand of the replication
fork. It contacts the first 21-30 nucleotides of the strand that
passes through its interior and the first 6-10 nucleotides of the
strand that does not pass through its center.
|
|
A second model is also possible. In this case, the 3'-single stranded
tail would pass through the central hole of the protein ring, and the
outside of the protein would then contact the 5'-single-stranded region
(Fig. 9B). This model would position the hexamer around the
leading strand of the replication fork. For T. aquaticus
DnaB, the 21-30 nucleotides of the strand that passes through the
protein, the 3'-tail, would now contact the helicase. The outside of
the hexamer would contact 6-10 nucleotides of the 5'-tail, which
corresponds to the lagging strand.
In this second model, the length of leading-strand DNA passing through
and contacting the T. aquaticus DnaB protein, 21-30 nucleotides, is consistent with the single-stranded DNA length bound by
E. coli DnaB, 20 ± 3 nucleotides (34). However, this model may contradict previously proposed models of 5' to 3' movement polarity for members of this helicase family. This model also contradicts the report based upon fluorescence data that the 5' single-stranded tail region of a forked DNA substrate passes through the inner channel of E. coli DnaB (37). This apparent
discrepancy can be explained if the orientation of the DnaB protein is
reversed in these fluorescence experiments, such that the protein is
positioned to move away from the duplex region. The ability of DnaB to
encircle the 3'-single stranded tail region of a forked DNA substrate
has not yet been assessed.
Because all polymerases have 5' to 3' polarity of synthesis, primase
must use the lagging strand as a template to initiate each cycle of
Okazaki fragment synthesis. For T7 phage, the gene 4 protein is
responsible for both helicase and primase activities. For E. coli, DnaB helicase binds to DnaG primase with weak affinity (38).
Thus, in either system, the replication fork helicase interacts with
the primase. In the first model presented above, the helicase is
encircling the template strand for primase, whereas in the second
model, the helicase is surrounding the opposite strand (Fig. 9). Thus,
the interaction between primase and helicase will be predominantly
same-strand in the first model and cross-strand in the second model.
Either model is possible, because it has recently been shown that the
T7 gene 4 protein can use either the strand that it is bound to or a
foreign strand as a template for priming (39).
While this work was in progress, two studies performing similar
experiments with the gene 4 protein from T7 phage were reported (15,
17). In these studies, the tail length dependence on unwinding rate is
different from that presented here for DnaB. These studies show
that ~ 35 nucleotides of the 5'-tail stimulate unwinding,
whereas only ~10-15 nucleotides of the 3'-tail stimulate unwinding.
Thus, the tail length asymmetry observed for the gene 4 protein is
nearly the reverse of that shown here for T. aquaticus DnaB.
It is likely that these two proteins unwind duplex DNA with a similar
mechanism because they are homologous. However, we are led to wonder
whether it is possible that the two related helicases encircle
different strands but still retain the same overall mechanism, which
would result in the 5'-tail passing through the center of T7 gene 4 protein, as in the first model presented above, and the 3'-tail passing
through the center of T. aquaticus DnaB, as in the second
model presented above. Alternatively, each of these proteins may
surround the same single-stranded tail at the replication fork, but the
specific DNA contact may be different. Future structural studies will
help determine which of these hypotheses is correct.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Yousif Shamoo and Satwik
Kamtekar for comments on the manuscript. We also thank Dr. Anna Lee
for help in preparing Fig. 9.
 |
Note Added in Proof |
Since submitting this manuscript for
review, we have performed unwinding assays using forked duplex DNA
substrates that are composed of oligonucleotides of mixed polarity. We
found that DnaB from T. aquaticus unwinds forked duplex DNA
containing two 5' single-stranded regions at an equal rate compared to
the standard substrate. By contrast, the enzyme could not unwind forked
duplex DNA containing two 3' single-stranded tails. These new
observations suggest that the generally accepted model shown in Fig.
9A is more likely than the alternative model illustrated in
Fig. 9B.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Institutes of Health.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF100420.
§
Howard Hughes Medical Institute Predoctoral Fellow.
**
To whom correspondence should be addressed: Dept. of Molecular
Biophysics and Biochemistry, Yale University, 266 Whitney Ave., New
Haven, CT 06520-8114. Tel.: 203-432-5617; Fax: 203-432-3282.
 |
ABBREVIATIONS |
The abbreviations used are:
PCR, polymerase
chain reaction;
GTP
S, guanosine-5'-(
-thio)triphosphate;
AMP-PNP, adenosine 5'-(
,
-imino)triphosphate;
AMP-PCP, adenosine
5'-(
,
-methylene)triphosphate;
ATP
S, adenosine-5'-(
-thio)triphosphate;
Mes, 2-(N-morpholino)ethanesulfonic acid;
DTT, dithiothreitol;
Bicine, N,N-bis(2-hydroxyethyl)glycine.
 |
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