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INTRODUCTION |
The first DNA helicase was identified and characterized more than
20 years ago (1, 2). Since then, the biochemical properties and
biological functions of many helicases have been firmly established (3-5), and new helicases are continually being discovered and characterized from prokaryotic, eukaryotic, and viral systems. Despite
the great attention helicases have received in recent years due to
their fundamental importance in DNA and RNA metabolism, many details of
the mechanism(s) by which helicases couple the energy derived from
nucleoside 5'-triphosphate hydrolysis to the separation of the two
strands of duplex nucleic acids are still not known.
Several models for helicase-catalyzed unwinding have been proposed
(reviewed in Ref. 5). A common feature of these models is the existence
of multiple DNA- or RNA-binding sites within the active enzyme.
Multiple binding sites are believed to be essential for processive
translocation of the helicase along the nucleic acid. This requirement
is thought to be satisfied by an oligomeric enzyme, and in general,
helicases have been divided into two oligomeric categories: dimers and
hexamers. Whereas the assembly state of several hexameric helicases has
been described in detail (6-12), information on dimeric helicases has
been mostly limited to studies of the E. coli Rep enzyme.
Lohman and colleagues (13-15), using a combination of steady-state and
pre-steady-state kinetic studies, have demonstrated that DNA binding
induces dimerization of E. coli Rep helicase and that dimers
are the active form of the enzyme. A model for processive DNA unwinding
catalyzed by the Rep helicase has been proposed in which the two
subunits of the active dimeric enzyme alternate binding to the
double-stranded DNA (dsDNA)1
region at an unwinding fork to catalyze ATP
hydrolysis-dependent strand separation (15). In this
rolling model, cycling of the two subunits through a duplex region
during processive unwinding is driven by changes in single-stranded
(ssDNA) and dsDNA binding affinities. These changes in affinity are
allosterically regulated by the state of nucleotide binding of each
subunit (recently reviewed in Ref. 5).
DNA helicase II (UvrD), which shares approximately 40% amino acid
sequence identity with Rep, performs a variety of functions in E. coli including essential roles in methyl-directed mismatch repair
(16) and nucleotide excision repair (17, 18). In a previous report,
UvrD was shown to form dimers and higher order oligomers in solution,
and dimerization was stimulated in the presence of ssDNA (19). It has
been proposed that UvrD functions as a dimer and may employ an
unwinding mechanism similar to that proposed for Rep (5, 20). This
suggestion is based on (i) the extensive sequence similarity between
Rep and UvrD, (ii) the abundance of data suggesting dimerization is
required for activity of the E. coli Rep helicase, (iii) the
observation that UvrD forms dimers and higher order oligomers in
solution, and (iv) current models for helicase-catalyzed DNA unwinding
mechanisms in which the requirement for multiple DNA-binding sites is
generally satisfied by multiple subunits in an active enzyme. However,
there is currently no direct evidence to indicate that UvrD is
functional as a dimer or to favor a rolling model for UvrD-catalyzed unwinding.
During the course of a yeast two-hybrid screen of an E. coli
genomic library using the uvrD gene as bait, we found that
UvrD interacts with itself in support of the notion of dimerization. However, a UvrD mutant was constructed that failed to dimerize yet
functioned in two DNA repair pathways in vivo and was active as a ssDNA-stimulated ATPase and a helicase in vitro. This
result prompted a thorough biophysical and biochemical analysis of the oligomeric state of UvrD. We conclude that UvrD is active as a monomer,
and unwinding mechanisms based on a monomeric helicase are discussed.
 |
EXPERIMENTAL PROCEDURES |
Materials
Bacterial Strains and Plasmids--
E. coli BL21(DE3)
(F
ompT [lon]
hsdSBrB-mB- gal
dcm
DE3) was from Novagen, Inc. BL21(DE3)
uvrD and
JH137
uvrD were constructed previously in this laboratory
(21). JH137 (K91 din D1:: MudI (Aprlac)) was obtained from P. Model.
Plasmids pET-9d, pET-11d, and pLysS were from Novagen, Inc. M13mp7
ssDNA was prepared as described (22). Construction of plasmids that
express UvrD and the various UvrD mutants have been described
previously (21, 23, 24). To construct a plasmid that expressed
UvrD
40C, pET11d-UvrD was digested to completion with
BsiW1. The 5' extension was filled in using DNA polymerase I
(large fragment) and dNTPs, and the plasmid was ligated with T4 DNA
ligase. This caused a +1 frameshift at codon 676, changing 5'-GTACGCCACGCTAAGTTT-3' (Val-Arg-His-Ala-Lys-Phe) to
5'-GTACGTACGCCACGCTAA-3' (Val-Arg-Thr-Pro-Arg-TER). The
uvrD
40C mutation was confirmed by DNA sequencing using
the Sequenase kit (U. S. Biochemical Corp.).
Oligonucleotides, Nucleotides, and Proteins--
Oligonucleotide
(dT)16 was from The Midland Certified Reagent Co. The
2-aminopurine (2-AP)-substituted oligonucleotides were synthesized by
Genosys. All nucleotides were from Amersham Pharmacia Biotech. All
enzymes used for cloning and PCR were from New England Biolabs, with
the exception of T4 DNA ligase, which was from Roche Molecular
Biochemicals. Thyroglobulin, catalase, rabbit muscle aldolase, and
cytochrome c were from Sigma. Human transferrin was from Calbiochem.
Protein Purification--
UvrD and UvrD
40C were overexpressed
prior to purification by growing either a 10-liter culture of
BL21(DE3)/pLysS cells containing pET11d-UvrD (21) or
BL21(DE3)
uvrD/pLysS cells containing pET11d-UvrD
40C to
an optical density (600 nm) of 1.0 at 37 °C. Protein expression was
induced by adding 0.4 mM
isopropyl-
-D-thiogalactopyranoside, and growth was
continued for an additional 4 h. Purification of wild-type
helicase II was performed according to a previously published procedure
(19). UvrD
40C was purified using the same procedure with one
modification. UvrD
40C was loaded onto an ssDNA-cellulose column (5.8 mg ssDNA/g of cellulose) at 0.1 M NaCl in Buffer A (20 mM Tris-HCl (pH 8.3 at 25 °C), 20% glycerol (v/v), 1 mM EDTA, 0.5 mM EGTA, and 15 mM
2-mercaptoethanol) instead of Buffer A + 0.2 M NaCl. The
column was washed using Buffer A + 0.2 M NaCl and eluted
using Buffer A + 1 M NaCl.
Methods
Yeast Two-hybrid System--
Plasmids and strains for the yeast
two-hybrid system were from CLONTECH. An E. coli genomic library was constructed previously in pGAD424 (25).
The library was screened for UvrD-interacting proteins as described
(25). The bait plasmid was pGBT9-UvrD. Vent DNA polymerase was used to
amplify the uvrD gene by PCR using pET9d-UvrD as target.
Amplified uvrD was cloned into the SmaI site of
pGAD424 and pGBT9 to create in-frame translational fusions with the
GAL4 transcriptional activation domain and DNA binding domain, respectively. These constructs were designated pGAD424-UvrD and
pGBT9-UvrD.
Deletions from the N and C termini of UvrD were generated using
convenient restriction enzyme sites within the uvrD gene. The restriction enzymes XmnI, AvaII, and
BstBI were used to generate the N-terminal deletions
uvrD
276N, uvrD
309N, and uvrD
383N, respectively. The uvrD PCR product described in the
preceding paragraph was digested individually with each of these
enzymes, and the appropriate fragment was purified and ligated into the SmaI site of pGAD424 to generate a fusion with the
GAL4 activation domain. Blunt ends were generated by a DNA
polymerase I (large fragment)-catalyzed fill-in reaction where
necessary. All clones produced in-frame translational fusions and were
confirmed by sequence analysis. pGAD424-UvrD
40C was generated by
digestion of pGAD424-UvrD with BsiWI and BglII,
gel purification, fill-in of the 5'-overhanging ends with DNA
polymerase I (large fragment), and re-ligation of the blunt ends.
Two-hybrid interactions were characterized using the lacZ
reporter gene in strain SFY526 or the HIS3 reporter gene in
strain HF7c. In HF7c, interactions were identified by growth on media lacking histidine. Assays for
-galactosidase activity encoded by the
lacZ gene in SFY526 were performed using the substrate o-nitrophenyl
-D-galactopyranoside as
described by the supplier (CLONTECH), and
quantified as Miller units (26).
Analytical Ultracentrifugation--
Sedimentation equilibrium
and sedimentation velocity experiments were done using a Beckman XL-A
centrifuge and an An-60ti rotor at 20 °C. Protein was prepared for
these experiments by extensive dialysis into the appropriate buffer.
For sedimentation equilibrium experiments the buffer contained 20 mM Tris-HCl (pH 8.3 at 25 °C), 0.2 M NaCl,
20% glycerol (v/v), 1 mM EDTA, 1 mM EGTA, and
15 mM 2-mercaptoethanol. For velocity sedimentation experiments the buffer contained 25 mM Tris-HCl (pH 7.5 at
25 °C), 50 mM NaCl, 3 mM MgCl2,
20% glycerol (v/v), and 5 mM 2-mercaptoethanol. Solvent
densities (
) were measured using a Mettler DA-110 M
density meter. The partial specific volume for UvrD and UvrD
40C was
calculated to be
= 0.729 at 20 °C using SEDNTERP (27).
Equilibrium ultracentrifugation experiments were performed in 6-channel
(1.2 cm path) charcoal-epon centrifuge cells (Beckman). The equilibrium
experiment was performed using three different concentrations of UvrD
(1.1, 2.1, and 3.8 µM) and UvrD
40C (2.8, 5.0, and 8.4 µM). Protein concentrations were determined from absorbances collected during the low speed scans in the
ultracentrifuge. Scans at 280 nm were recorded every 2 h. After
30 h the cell was judged to be at equilibrium. This was confirmed
by subtracting successive plots and examining residuals for the absence
of any systematic patterns. For UvrD the speeds used were 5,000, 7,500, and 10,000 rpm, and for UvrD
40C the speeds were 7,200, 8,600, and
12,600 rpm. All concentrations of UvrD
40C reached equilibrium within
the 30-h period for each speed. Three of the data sets obtained with
the wild-type protein did not reach equilibrium during the course of
the experiment (1.1 µM at 5,000 rpm, 2.1 µM
at 5,000 rpm, and 3.8 µM at 10,000 rpm) and were not
considered in the analysis.
Several models were fit to data from equilibrium centrifugation
experiments using NONLIN (28). Separate concentrations and different
speeds were analyzed individually, in groups, and globally. The models
primarily considered were single ideal species and ideal monomer
dimer equilibrium. In addition, other models were examined including
non-ideality, monomer
trimer and monomer
tetramer. Molecular
masses were calculated based on the values for
(reduced molecular
mass) (see Equation 1) according to methods described (28).
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(Eq. 1)
|
M is the molecular weight; R is the gas
constant; T is the temperature in degrees Kelvin;
is the
partial specific volume (ml/g) of the protein;
is the angular
velocity of the rotor (radians/s), and
is the measured density of
the buffer (g/ml). Kd values were calculated using
Equation 2 for the model of monomer
dimer equilibrium (see Equation 3).
|
(Eq. 2)
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|
(Eq. 3)
|
C(r) is the total protein concentration
(mg/ml) at the radius (r);
is the base-line offset used
to compensate for absorbance measurements of 0 not being precisely
equal to 0 protein concentration; C1,0 is the
monomer concentration at the reference distance
(r0); and K2 is the
association constant for monomer
dimer model (28, 29). The
reference distance, or reference radial position, is chosen in data
analysis and is usually near the meniscus in the cell.
1
is the reduced molecular weight for the monomer.
Velocity experiments were performed with 350-µl samples in
two-channel charcoal epon centerpieces (1.2 cm path) (Beckman). The
rotor speed was 50,000 rpm. Initial protein concentrations were
approximately 7.5 µM. Experimental samples analyzed in
the presence of nucleotide contained 0.2 mM AMP-PNP. For
reactions containing ssDNA, the DNA molecule used was a
(dT)16 oligonucleotide containing the modified base
2-aminopurine (AP). The two ssDNA substrates were either
5'-TTTTT(AP)TTTT(AP)TTTTT-3' or 5'-TTT(AP)TT(AP)TT(AP)TT(AP)TTT-3'. The molar ratio of protein to DNA was 1:1.25. Data obtained from sedimentation velocity experiments in the absence of DNA (protein alone
or protein with nucleotide) were collected at either 285 or 290 nm. In
the presence of DNA, sedimentation was monitored at 315 nm to detect
only the modified (dT)16 molecule. Scans were recorded at
1-min intervals in a continuous mode.
Sedimentation velocity data were analyzed using the program Svedberg
(30). Sedimentation coefficients (s*) were determined by
fitting the modified Fujita-MacCoshman equation for a single species
and two species to the data (31). For experiments containing DNA, the
program dCdT (32) was used to obtain sw values for
interacting species. The sw values obtained using dCdT were consistent with s* values obtained using Svedberg.
A control sedimentation velocity experiment performed using the 2-AP
oligonucleotide in the absence of protein confirmed the identity of the
two species in the experiment containing DNA (DNA alone and DNA-protein
complex). The values s* and sw were
corrected for solvent density and viscosity and normalized to standard
conditions to produce the s20,w value
(33, 34). Values for
were corrected for percentage glycerol as
described (35, 36).
Gel Filtration--
The apparent molecular mass of UvrD and UvrD
mutants was determined in the presence and absence of ligands using a
Superose 12 HR 10/30 column (Amersham Pharmacia Biotech) and high
pressure liquid chromatography system (Rainin, model HPXL) at 4 °C.
Column buffer was 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl2, 5 mM 2-mercaptoethanol, and 20% glycerol. When present in
the column buffer, ATP was 0.5 mM. Proteins (25 µg) were
injected onto the column in a volume of 50 µl at a flow rate of 0.2 ml/min. When present, 32P-labeled oligonucleotide
(dT)16 was included in the loaded sample at a concentration
of 11.2 µM. Samples were passed through the column at a
flow rate of 0.2 ml/min, and elution of protein peaks was monitored
continuously by absorbance at 280 nm. The elution of (dT)16
was followed by scintillation counting of column fractions. Samples
were subjected to centrifugation prior to loading to remove particulate
matter and any insoluble protein. Comparison of protein concentration
measurements made before and after application to the column revealed
that essentially all protein was soluble and recovered in a single
peak. A standard curve of log molecular mass versus
retention time was generated in the absence of ATP using the following
proteins under the column buffer conditions described above:
thyroglobulin (668,000 Da), catalase (212,000 Da), rabbit muscle
aldolase (158,000 Da), human transferrin (80,000 Da), and cytochrome
c (12,400 Da). The retention time of all protein standards
was not significantly altered by the presence of ATP.
Genetic Assays--
Genetic complementation assays were
performed using JH137 and derivatives. UV irradiation survival assays
(23) and determination of spontaneous mutation frequencies (37) were
performed as described previously.
ATPase Assays--
The standard ATPase reaction mixture
contained 25 mM Tris-HCl (pH 7.5), 3 mM
MgCl2, 20 mM NaCl, and 5 mM
2-mercaptoethanol. The reaction stop solution was 33 mM
EDTA, 7 mM ATP, and 7 mM ADP. In general,
reactions were executed and products separated by thin layer
chromatography as described previously (38). All reactions were
incubated at 37 °C. When [3H]ATP was used, the results
were quantified using a liquid scintillation counter. When
[
-32P]ATP was used, the results were quantified using
a Storm 840 PhosphorImager and ImageQuant software (Molecular
Dynamics). Background signals measured in the absence of enzyme were
typically between 1 and 2% and were subtracted from the experimental data.
For inhibition assays the concentration of UvrD was 4 nM
and the concentrations of UvrD-K35M, UvrD-E221Q, and UvrD-R605A were 125 nM. Reaction mixtures (30 µl) contained 1 mM [
-32P]ATP and 1.8 µM
oligonucleotide (dT)16 (molecules). Wild-type and mutant
helicase II enzymes were mixed together at 15 times their final
concentrations and preincubated on ice for 10 min. Reactions were
initiated by adding enzyme (wild-type, mutant, or a mixture of both) to
reaction mixtures at 37 °C. Samples (5 µl) were removed at 1-min
intervals (within the linear range of the reaction) and quenched with 5 µl of stop solution.
Reactions for examining the protein concentration dependence of the
UvrD-catalyzed ATPase reaction were identical to those for the
inhibition assays except that the UvrD concentration was varied from 1 to 64 nM, and bovine serum albumin was included at 50 µg/ml. As the UvrD concentration was decreased, samples were removed
at increasing time intervals to ensure production of a detectable
signal. All data points fell within the linear range of the reaction.
To compare the kcat values of UvrD and
UvrD
40C, reaction mixtures (30 µl) contained 0.8 mM
[3H]ATP and 30 µM M13mp7 ssDNA (nucleotide
phosphate). Reactions were initiated with enzyme at a final
concentration of 2 nM. Samples (8 µl) were removed and
quenched with 8 µl of stop solution at 2-min intervals. To compare
Km values of UvrD and UvrD
40C, reaction mixtures
(20 µl) contained 2 nM enzyme, 30 µM M13mp7 ssDNA (nucleotide phosphate), and increasing concentrations (25-500 µM) of [
-32P]ATP. Reactions were
initiated with [
-32P]ATP and incubated for 10 min.
Duplicate samples of 5 µl were removed and quenched with 5 µl of
stop solution.
Helicase Assays--
Helicase reaction mixtures (20 µl)
contained 25 mM Tris-HCl (pH 7.5), 3 mM
MgCl2, 20 mM NaCl, 5 mM
2-mercaptoethanol, and 3 mM ATP. For inhibition
experiments, the reaction mixtures also included 0.5 µM
20-mer oligonucleotide molecules and 5 nM 20-bp partial
duplex [32P]DNA substrate molecules. The partial duplex
substrate was prepared as described previously (23). The unlabeled
20-mer was the same sequence as the 32P-labeled 20-mer used
to make the partial duplex substrate and was included at molar excess
to prevent reannealing of unwound 32P-labeled 20-mer. The
concentration of UvrD was 1 nM, and the concentrations of
UvrD-K35M, UvrD-E221Q, and UvrD-R605A were 3 nM. Wild-type
and mutant helicase II enzymes were mixed together at 10 times their
final concentrations and incubated on ice for 10 min. Subsequently,
enzymes were incubated on ice with the partial duplex DNA substrate in
the reaction buffer for 5 min prior to initiation of the reactions.
Reactions were initiated by adding ATP and unlabeled 20-mer at
37 °C. After incubation at 37 °C for 3 min, reactions were
quenched with 10 µl of stop solution (37.5% glycerol, 50 mM EDTA, 0.05% each of xylene cyanol and bromphenol blue,
and 0.3% SDS).
For the biochemical comparison of UvrD- and UvrD
40C-catalyzed
unwinding reactions, the standard reaction mixtures included approximately 0.2 nM 92-bp partial duplex
[32P]DNA substrate molecules prepared as described
previously (23). Reactions were initiated with the indicated
concentration of enzyme at 37 °C, incubated for 10 min, and quenched
with 10 µl of stop solution. All reaction products were resolved on
8% non-denaturing polyacrylamide gels (20:1 cross-linking ratio), and
the results were visualized and quantified using a Storm 840 PhosphorImager and ImageQuant software (Molecular Dynamics).
 |
RESULTS |
UvrD Interacts with Itself in the Yeast Two-hybrid
System--
The formation of dimers and higher order oligomers by UvrD
has been suggested based on gel filtration techniques and
glutaraldehyde cross-linking (19). This observation is consistent with
the idea that the active form of UvrD may be dimeric, as has been demonstrated for Rep helicase (13-15). We discovered independent evidence for UvrD dimerization that enabled us to begin to identify the
domain(s) responsible for this phenomenon.
In a yeast two-hybrid screen to identify proteins that interact with
UvrD, an interacting clone was isolated that was identical to a portion
of the uvrD gene. This interacting clone encoded a region of
UvrD lacking the N-terminal 244 amino acids, suggesting that the N
terminus of helicase II was not essential for oligomerization. Subsequently, an interaction between two full-length UvrD proteins was
demonstrated (Fig. 1A). To
define further the interaction domain, various deletion mutants were
constructed and analyzed for their ability to interact with wild-type
UvrD in the yeast two-hybrid system using assays for
-galactosidase
activity to quantify interaction-dependent expression of a
lacZ reporter gene (Fig. 1B). The results
indicated that the C-terminal half of UvrD was sufficient to produce a
detectable interaction. Removal of 383 amino acids from the N terminus
or 40 amino acids from the C terminus abolished the two-hybrid
interaction. The latter result was particularly interesting because the
UvrD
40C mutant had been partially characterized previously and found
to be indistinguishable from wild-type
UvrD.2 The two-hybrid data
suggest that UvrD
40C has a defect in oligomerization, which prompted
a careful examination of this property using both UvrD and UvrD
40C.
The existence of a mutant that fails to dimerize, yet has wild-type
biochemical activity, has significant implications for the
UvrD-unwinding mechanism.

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Fig. 1.
UvrD interacts with itself in the yeast
two-hybrid system. A, HF7c cells containing pGAD424 and
pGBT9 with or without the uvrD gene were grown at 30 °C
on complete synthetic media lacking tryptophan, leucine, and histidine
and supplemented with 1 mM 3-amino-1,2,4-triazole. Each
quadrant contains cells streaked from a single transformant that was
colony-purified. Labels represent fusion proteins present in the HF7c
cells in the order, DNA binding domain fusion/transcriptional
activation domain fusion. A represents the absence of
uvrD from the fusion construct. B,
-galactosidase activity was measured in yeast SFY526 cells using
o-nitrophenyl -D-galactopyranoside as
described under "Experimental Procedures." Truncations of the
uvrD gene were constructed in pGAD424 and were tested for an
interaction in the presence of pGBT9-UvrD. A + indicates the presence
of an interaction and a indicates the absence of an
interaction. All assays scored as interactions displayed at least a
40-fold increase in -galactosidase activity (Miller units) compared
with a control in the absence of a UvrD-activation domain fusion. All
results represent the average of 2 or 3 separate experiments using
independent transformants.
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Assembly State of UvrD and UvrD
40C--
The assembly state
(i.e. monomer, dimer, oligomer) of UvrD and UvrD
40C was
examined by analytical sedimentation equilibrium ultracentrifugation
experiments (Fig. 2). The predicted
molecular masses, based on amino acid composition, are 82,151 Da for
UvrD and 77,850 Da for UvrD
40C. The average apparent molecular mass, as revealed by equilibrium sedimentation, was 119.8 kDa for UvrD and
61.1 kDa for UvrD
40C. These apparent molecular masses represent the
average mass of species present in the ultracentrifuge cell.

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Fig. 2.
Sedimentation equilibrium of UvrD and
UvrD 40C. The association state of UvrD
and UvrD 40C was analyzed using analytical ultracentrifugation. Data
shown are a subset of all the data acquired at three different speeds
and protein concentrations for both UvrD and UvrD 40C as described
under "Experimental Procedures." A, UvrD samples (2.1 µM) were sedimented at 7,500 rpm ( ) and 10,000 rpm
( ). The equation for the monomer dimer equilibrium (2) was fit
to the data (solid line). B, UvrD 40C (5.0 µM) samples were sedimented at 7200 ( ) and 12,960 rpm
( ). The monomer dimer model, using Equation 2 (solid
line) and the equation for a single ideal species,
C(r) = + C1,0e
( 1 ) (dashed line) (28), were fit to the
data. Protein absorbance was measured at 280 nm. Data analysis was
performed using NONLIN (28) as detailed under "Experimental
Procedures."
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|
For wild-type helicase II, molecular mass was consistent with a mixture
of monomers and dimers. Several models were fit to the equilibrium
centrifugation data for UvrD and UvrD
40C, including single ideal
species, monomer
dimer, monomer
trimer, monomer
tetramer,
and non-ideality. All of the data analyzed for UvrD were most
consistent with the monomer
dimer model described by Equation 2
(Fig. 2A, solid line). Using this equation, the Kd for dimerization was calculated to be 3.4 µM.
The average apparent molecular mass determined for UvrD
40C was
consistent with the monomeric molecular mass. In addition, the apparent
molecular mass of UvrD
40C did not increase with an increase in
protein concentration, even at protein concentrations more than 2-fold
greater than the highest wild-type protein concentration. The lower
than expected apparent molecular mass for UvrD
40C may be due to the
presence of 20% glycerol in the ultracentrifuge cell. It has been
reported that glycerol can affect the observed molecular mass in
equilibrium sedimentation experiments (36). If this is the case, then
the molecular mass reported for UvrD may be an underestimate. All of
the data for UvrD
40C were most consistent with a model for a
single-ideal species (Fig. 2B, dashed line) with
a monomeric molecular mass. A dimerization constant for UvrD
40C
could not be determined since the monomer
dimer model failed to
converge to the data (Fig. 2B, solid line). Thus, UvrD
40C fails to dimerize, consistent with the results obtained from
the yeast two-hybrid system.
To determine whether ligands could enhance the dimerization of either
protein, sedimentation velocity experiments were performed in the
presence and absence of a poorly hydrolyzable ATP analog and ssDNA. It
has been shown previously, using other techniques, that dimerization of
Rep occurs only on ssDNA and is therefore ligand-induced (13, 15). It
is important to note that the sedimentation coefficient
(s20,w) reflects both the size and shape
of the protein. The s20,w of a dimer
should increase by a factor of 1.5 over that for a monomer (39, 40). Such an increase has been demonstrated previously for the gene 41 helicase from phage T4 which dimerizes in the presence of GTP (11).
Sedimentation velocity experiments with UvrD were possible only in the
presence of DNA and AMP-PNP, due to the limited solubility of wild-type
helicase II in the absence of DNA (Table
I). However, it was possible to measure
sedimentation velocity for UvrD
40C in the absence of ligands, in the
presence of nucleotide, and in the presence of DNA plus nucleotide.
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Table I
Sedimentation coefficients
Velocity sedimentation experiments were performed as described under
"Experimental Procedures." UvrD and UvrD 40C were approximately
7.5 µM. Data from experiments containing protein alone
and those with protein and AMP-PNP (0.2 mM) were collected
at 290 nm. Data from experiments containing the 2-AP-modified
(dT)16 oligonucleotide were collected at 315 nm. DNA to protein
molar ratios were 1.25:1. s* values for the protein alone
and protein plus AMP-PNP were obtained using Svedberg (30). Experiments
containing DNA were analyzed using the dCdT program, and
sw values were obtained based on the assumption of
interacting species (32). s* and sw
(uncorrected sedimentation coefficients) were normalized to standard
conditions (s20,w) as described under
"Experimental Procedures."
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The UvrD
40C sedimentation coefficient in the absence of ligands was
7.0 ± 0.3 (Table I). Scans of the ultracentrifuge cell during the
course of the velocity sedimentation experiments failed to show any
biphasic character suggestive of multiple species in the cell
(i.e. populations of monomers and dimers). Moreover, multiple species models did not fit the data as well as the modified Fujita-MacCoshman function for a single species. The sedimentation coefficient for UvrD
40C decreased in the presence of an ATP analog (AMP-PNP) relative to that of the protein alone and decreased further
in the presence of (dT)16 and AMP-PNP. The decrease in s20,w indicates that the protein is
undergoing a conformational change in the presence of these ligands.
The change in s20,w is not consistent
with the notion that the protein dimerizes in the presence of either
one or both ligands. Even if the assumption that this protein behaves
as an anhydrous sphere is incorrect, it is unlikely that the
sedimentation coefficient would decrease upon dimerization because
s20,w is directly proportional to the
molecular mass of the species and inversely proportional to shape
factors. The sedimentation coefficient for wild-type helicase II in the
presence of AMP-PNP and DNA was 5.1 ± 0.1. This was strikingly
similar to the result obtained for UvrD
40C under the same
conditions. It is known from the equilibrium sedimentation experiments
described above that UvrD
40C behaves as a monomer. Thus, this result
suggests that the monomeric form of wild-type UvrD may be stabilized in
the presence of an ATP analog and ssDNA.
UvrD
40C Elutes as a Monomer on a Gel Filtration Column--
To
confirm the UvrD
40C oligomerization defect revealed in the yeast
two-hybrid and ultracentrifugation experiments, high pressure liquid
chromatography gel filtration was used to determine the apparent
molecular mass of UvrD and UvrD
40C in the presence and absence of
ligands. In the absence of ligands, UvrD eluted as a single peak with
an apparent molecular mass between that expected for a monomeric (82 kDa) and dimeric (164 kDa) protein (Fig.
3A and Table
II). Under very similar solution
conditions (specifically the presence of Mg2+) Runyon
et al. (19) observed the same result, and it is consistent with a rapid equilibrium between monomeric and oligomeric species. In
contrast to UvrD, UvrD
40C eluted as a single peak consistent with
the predicted molecular mass for the monomeric protein (78 kDa). It
should also be noted that the UvrD elution peak was consistently broader than that of UvrD
40C and exhibited a shallow trailing slope,
suggesting that UvrD existed as a heterogeneous population of molecules
(Fig. 3, A and B). The symmetry of the UvrD
40C
peak is consistent with a homogeneous population of molecules.

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Fig. 3.
UvrD and UvrD 40C
elute from a Superose 12 column at different positions. 25 µg of
UvrD or UvrD 40C were applied to a Superose 12 column as described
under "Experimental Procedures" in the absence (A) and
presence (B) of 500 µM ATP. The retention
times of the samples were defined as the points of maximum absorbance
at 280 nm on the elution traces and are indicated by the vertical
dashed lines. Independent traces of UvrD and UvrD 40C were
superimposed on one another. Arrows indicate the elution
peaks of the five proteins used to generate the standard curve for
apparent molecular mass calculations. B, the absorbance at
280 nm was initially normalized to zero to eliminate the contribution
of ATP to the signal. This resulted in the lower apparent absorbances
of UvrD and UvrD 40C compared with those in A.
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In the presence of ATP, UvrD eluted with a lower apparent molecular
mass than in the absence of ATP (Fig. 3B and Table II) but
still appeared to exist in a rapid equilibrium between monomeric and
dimeric species. The apparent molecular mass of UvrD
40C did not
change significantly in the presence of ATP, again suggesting that this
protein exists in solution as a monomer. The shift in the UvrD elution
peak is consistent with the notion that ATP binding stabilizes the
monomeric form of UvrD. The shift to a lower apparent molecular mass is
not consistent with an ATP-induced dimerization of UvrD.
To investigate the possibility that DNA binding affects the oligomeric
state of UvrD or restores the ability of UvrD
40C to dimerize, gel
filtration was performed using a pre-formed enzyme-ssDNA complex. In
the presence of ATP and oligonucleotide (dT)16, the apparent molecular masses of UvrD and UvrD
40C increased by
approximately the same amount compared with experiments in which only
ATP was present (Table II, 119 versus 98 kDa for UvrD and 91 versus 73 kDa for UvrD
40C). Since (dT)16
eluted from the column with an apparent molecular mass of 26 kDa (Table
II), the size of the increase in each case was consistent with that
expected from binding of (dT)16 to the enzyme and was not
large enough to suggest a stimulation of oligomerization. It is
important to note that the apparent molecular mass of the
UvrD
40C-(dT)16 complex was still significantly lower
than that of the UvrD-(dT)16 complex. Thus, the
oligomerization defect exhibited by UvrD
40C in the absence of DNA
was not corrected by the presence of ssDNA. We confirmed that the
protein-(dT)16 complex was reasonably stable during the gel
filtration experiments by using radiolabeled (dT)16 and
comparing the elution positions of the DNA and protein (data not
shown). Although the protein-ssDNA complexes were clearly in a rapid
equilibrium between bound and unbound states relative to the time
course of the experiments, the concentration of (dT)16 in
the protein peak indicated that greater than 60% of the helicase II
was bound to DNA.
Genetic and Biochemical Characterization of UvrD
40C--
Since
UvrD
40C fails to oligomerize, and oligomerization has been suggested
to be essential for helicase activity (5), it was of interest to
evaluate the activity of UvrD
40C in genetic and biochemical assays.
The ability of UvrD
40C to complement the loss of UvrD in
methyl-directed mismatch repair and excision repair was examined using
a strain lacking the uvrD gene. JH137
uvrD was
transformed with pET9d-UvrD
40C and pET9d-UvrD. Uninduced expression
of UvrD and UvrD
40C from these constructs in JH137
uvrD was detectable by Western blot and was similar to chromosomal levels of
expression of the wild-type gene in JH137 (data not shown). To assess
function in methyl-directed mismatch repair, the spontaneous mutant
frequency was measured by quantifying the number of spontaneously
arising rifampicin-resistant colonies. Previous studies have shown that
wild-type UvrD, expressed from a plasmid, fully complements the loss of
helicase II (23, 37, 41). The relative mutability of
JH137
uvrD was 240-fold greater than the parental strain,
JH137 (data not shown). JH137
uvrD containing
pET9d-UvrD or pET9d-UvrD
40C exhibited relative mutant frequencies of
1.01 and 0.92, respectively, demonstrating complete complementation of
the uvrD deletion (Table III). The UV sensitivity of these
strains was also measured at increasing doses of UV irradiation to
evaluate nucleotide excision repair function. The UV sensitivity of
JH137
uvrD/pET9d-UvrD
40C was comparable to that of
JH137 and JH137
uvrD/pET9d-UvrD (data not shown). Thus,
UvrD
40C is fully functional in both mismatch and excision repair.
UvrD
40C was also characterized in biochemical assays. UvrD and
UvrD
40C were purified to apparent homogeneity (data not shown), and
UvrD
40C was assayed for ssDNA-stimulated ATPase activity and DNA
helicase activity. The ssDNA binding ability of UvrD
40C was
investigated previously and was found to be similar to that of the
wild-type protein.2 The turnover rates for ssDNA-stimulated
ATP hydrolysis (kcat) for both proteins were not
significantly different (147 versus 157 s
1).
In addition, both UvrD
40C and UvrD appeared to interact with nucleotide with the same affinity as evidenced by the nearly identical Km values for ATP (62 versus 50 µM). The helicase reaction catalyzed by each protein was
measured using a 92-bp partial duplex DNA substrate (Fig.
4). UvrD and UvrD
40C unwound the
partial duplex substrate with equivalent efficiency. Similar results
were obtained with a 238-bp blunt duplex substrate (data not shown). The rates of unwinding of the 92-bp partial duplex substrate by UvrD
and UvrD
40C during the course of a 10-min reaction were similar. In
fact, the rate of unwinding catalyzed by UvrD
40C was reproducibly
slightly greater than that of UvrD (data not shown). Thus, UvrD
40C
possesses wild-type ATPase and helicase activities in vitro,
consistent with its ability to function in vivo.

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Fig. 4.
Helicase activity of UvrD and
UvrD 40C. The unwinding activity of UvrD
( ) and UvrD 40C ( ) was measured using a 92-bp partial duplex
substrate as described under "Experimental Procedures." The
fraction of unwound substrate molecules was calculated for each protein
concentration as outlined (24). Data represent the average of at least
3 independent experiments, and error bars are standard
deviations about the mean. Continuous lines connecting data
points were drawn using a cubic spline algorithm (SigmaPlot).
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The ssDNA-stimulated ATPase Activity of UvrD Is Independent of
Protein Concentration--
Since UvrD
40C fails to oligomerize and
exhibits wild-type biochemical and genetic activity, we suggest that
the protein is functional as a monomer. This prompted further
investigations to determine if wild-type UvrD was also functional as a
monomer. Toward this end, several biochemical properties of UvrD were evaluated.
The kcat for ssDNA-stimulated ATP hydrolysis
catalyzed by UvrD was previously reported to increase by a factor of
2.5 as a function of enzyme concentration between 2 and 10 nM (19). Thus, the rate of hydrolysis of UvrD was
non-linearly dependent on enzyme concentration in this range. This
result was interpreted as evidence for the dimerization of UvrD causing
a stimulation of its ATPase activity and as support for an unwinding
model involving an active dimeric enzyme. Because the stimulation of
ATPase activity was small (only 2.5-fold) and UvrD still demonstrated
significant activity at concentrations below the inflection point, we
attempted to reproduce this result. Under our reaction conditions, the
ssDNA-stimulated ATPase activity of UvrD was independent of protein
concentration between 1 and 64 nM. In other words, the rate
of ATP hydrolysis was linearly dependent on UvrD concentration (Fig.
5) and provided no evidence for a change
in assembly state. Since typical biochemical DNA unwinding assays of
UvrD are performed within this range of protein concentrations, these
results must be representative of the active species. Unless UvrD has a
dimerization constant below 1 nM under these conditions
(which we have demonstrated is not the case), these results strongly
argue that UvrD monomers are an active form of the enzyme, at least as
an ATPase.

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Fig. 5.
The UvrD-catalyzed ssDNA-stimulated ATPase
reaction is linearly dependent on enzyme concentration between 1 and 64 nM. ATPase assays were performed at 37 °C as
described under "Experimental Procedures" using oligonucleotide
(dT)16 as the ssDNA effector. The straight line
is a linear regression (SigmaPlot) and corresponds to a
kcat of 78 s 1. The data for each
enzyme concentration were determined in individual experiments
involving 5 time points, and each data point represents the average of
2 separate trials.
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ATPase- and Helicase-deficient UvrD Mutants Do Not Inhibit the
ssDNA-stimulated ATPase or ATP-dependent Helicase
Activities of Wild-type UvrD--
As a further test of our conclusion
that UvrD monomers are active as DNA helicases, the effect of adding
non-functional UvrD mutants to ATPase and helicase reactions was
evaluated. If a dimeric enzyme were required for biochemical activity,
then the presence of an excess molar concentration of an inactive
mutant should inhibit the reaction catalyzed by the wild-type enzyme,
assuming random association of wild-type and mutant subunits. Such a
result has been observed previously with the E. coli Rep
helicase (42) and the bacteriophage T7 gene 4 helicase-primase (43, 44) which are known to function as a dimer and hexamer, respectively. The
ssDNA-stimulated ATPase reaction catalyzed by UvrD was measured in the
presence of three UvrD point mutants that were severely compromised for
ATPase activity (Fig. 6A).
Each mutant contained a single amino acid substitution of a highly
conserved residue within one of the helicase motifs and had been
characterized previously (21, 23, 24). Mutant enzymes were present at a
30-fold molar excess over wild-type UvrD, and oligonucleotide
(dT)16 was used as the ssDNA effector at a molar excess
over enzyme to ensure that ssDNA availability was not limiting.
Wild-type and mutant enzymes were co-incubated under conditions that
favor monomeric species prior to initiation of the reactions to ensure
random mixing of protein molecules. At this molar excess of mutant
protein, essentially all wild-type molecules should be complexed as a
heterodimer with an inactive mutant if dimers are formed. The results
clearly demonstrate that the presence of a molar excess of mutant
enzyme did not significantly inhibit the ssDNA-stimulated ATPase
activity of wild-type UvrD. Although the kcat
for ATP hydrolysis appeared to decrease slightly in the presence of
each mutant (112 versus 79-89 s
1), a
requirement for active dimers should have resulted in more dramatic
inhibition (to mutant kcat levels 0.058-0.283
s
1). Co-incubation of mutant and wild-type enzymes for a
much longer period prior to the reaction did not alter the results
(data not shown). In addition, co-incubation of the two proteins in the presence of Mg2+ or Mg2+-ATP or inclusion of
mutant enzyme at a 300-fold molar excess did not yield different
results (data not shown).

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Fig. 6.
Inactive UvrD point mutants do not inhibit
the ATPase or helicase activities of wild-type UvrD. A,
ATPase assays were performed as described under "Experimental
Procedures." When present, the concentration of UvrD was 4 nM, and the concentration of each mutant was 125 nM. Data represent the average of at least 3 independent
trials. When both wild-type and mutant enzymes were present, the
contribution of mutant enzymes to kcat was
subtracted from the data. B, helicase assays were performed
as described under "Experimental Procedures." When present, the
concentration of UvrD was 1 nM, and the concentration of
mutant enzymes was 3 nM. The concentration of 20-bp partial
duplex DNA substrate molecules in all reactions was 5 nM.
The data for wild-type or mutant enzymes alone are an average of at
least 3 independent trials. The data for reactions containing both
wild-type and mutant enzymes are an average of at least 5 independent
trials. The horizontal dashed line represents the expected
level of unwinding in reactions containing wild-type and mutant enzymes
if a dimer were the active species and assuming random association of
protein molecules. In both panels error bars are standard
deviations.
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It is possible to imagine a mechanism for dimer-mediated helicase
unwinding in which the two subunits of the dimer act independently as
ATPases. In such a scenario, inhibition of the ATPase reaction by
inactive mutant enzymes would not be observed. However, it is
considerably more difficult to imagine a dimer-mediated unwinding model
in which mutants did not inhibit unwinding of duplex DNA requiring
multiple turnovers of the enzyme.
Helicase inhibition assays similar to the ATPase inhibition assays
described above were performed, in which the unwinding activity of UvrD
was measured in the presence of the same catalytically compromised
mutant enzymes. Mutant enzymes were present at a 3-fold molar excess
over wild-type enzyme (higher concentrations of the mutant proteins
could not be achieved for technical reasons), and a 20-bp partial
duplex DNA substrate was present at a molar concentration slightly
greater than the total concentration of enzyme monomers. The step size
for UvrD-catalyzed DNA unwinding was recently reported to be 4-5
nucleotides (20). Therefore, displacement of the 20-mer oligonucleotide
should require 4-5 cycles of catalysis. Again, the wild-type and
mutant proteins were co-incubated prior to initiation of the reaction
(see "Experimental Procedures"). A large molar excess of unlabeled
20-mer was included upon initiation of the reaction with ATP to prevent
reannealing of displaced radiolabeled 20-mer molecules. This rendered
the reactions pseudo-single turnover. Under these conditions, UvrD alone unwound 25% of the DNA substrate (Fig. 6B). Unwinding
by the three mutant enzymes alone was negligible, as expected. Assuming random association of subunits, a 3:1 ratio of mutant to wild-type enzyme should result in 25% activity compared with wild-type UvrD alone if an active dimeric species were required for unwinding. Fig.
6B demonstrates that this result was not obtained. These results, coupled with the ATPase results, indicate that UvrD is functional as a monomer and that oligomerization is not obligatory for
catalytic competency.
Although the mutant enzymes could be defective at dimerization, it is
unlikely that three separate point mutations in different regions of
the protein would all impact oligomerization. However, to ensure that
this was not the case, the apparent molecular mass of the three mutant
enzymes was determined by gel filtration and compared with UvrD (see
Table II). All four proteins exhibited similar apparent molecular
masses that were suggestive of a rapid equilibrium between monomeric
and oligomeric species. In addition, the UvrD-K35M protein was analyzed
by sedimentation equilibrium ultracentrifugation, and the data were
described by a monomer
dimer equilibrium with a dissociation
constant similar to that of UvrD (data not shown). The average apparent
molecular mass for UvrD-K35M was 116.7 kDa, similar to the wild-type
value. These results suggest that the oligomerization properties of the
mutant enzymes were not compromised.
Non-functional Mutant UvrD Alleles Are Recessive to Wild-type UvrD
in Two DNA Repair Pathways--
Data obtained from previous genetic
studies are also consistent with the conclusion that UvrD is active as
a monomer. Site-directed mutagenesis of highly conserved residues in
the consensus helicase motifs resulted in the generation of point
mutants that failed to function in vivo (21, 23, 24, 37,
45). Although previously reported for several UvrD mutants, little
attention was directed to the recessive nature of all non-functional
alleles in a wild-type uvrD background. Table
III shows previously published genetic
complementation data for four uvrD alleles that lack
in vivo mismatch repair function. When expressed in
JH137
uvrD, none of the mutant alleles substituted for
wild-type UvrD (first data column). Furthermore, in JH137, containing a
wild-type uvrD allele on the chromosome, none of the mutant
alleles affected the spontaneous mutant frequency (second column)
indicating that the mutant alleles were recessive to the wild-type
gene. It should be noted that mutant alleles were expressed at near
chromosomal levels as evidenced by Western blots (data not shown). The
lack of a dominant negative phenotype is consistent with a model in
which UvrD acts as a monomer since the inactive mutant enzymes did not
interfere with function of the wild-type enzyme. Similar results were
obtained with various other uvrD alleles examined for
mismatch and/or excision repair function (data not shown). Efforts to
overexpress mutant uvrD alleles in a wild-type background to
observe a dominant negative phenotype were not interpretable since
overexpression of wild-type UvrD resulted in UV sensitivity and
increased mutation rate.
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Table III
Genetic complementation experiments
An expression plasmid encoding the indicated allele was transformed
into either JH137 uvrD or JH137. Spontaneous mutant
frequency was measured as described (37). Expression of mutant alleles
was confirmed by Western blot and was approximately equal to
chromosomal expression of the wild-type allele. Relative mutant
frequencies with respect to the parental strain, JH137, were obtained
by dividing the mutant frequency of each strain by that of JH137. ND,
not determined.
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DISCUSSION |
The precise mechanism by which a DNA helicase catalyzes the
unwinding of duplex DNA is not known, although it is clear that this
process requires energy supplied by the hydrolysis of NTPs. Thus, there
must be a coupling of ATP hydrolysis with disruption of the hydrogen
bonds between the two strands of duplex DNA. It has been postulated
that helicase-catalyzed unwinding requires an oligomeric enzyme (at
least a dimer), and reasonably detailed models have been proposed for
unwinding by a dimeric (15) or hexameric helicase (5, 46). A
fundamental component of these models is the notion that each protomer
in the oligomer contributes a DNA-binding site. Thus, an oligomer has
at least two DNA-binding sites allowing the enzyme to remain in contact
with the DNA, through one of these binding sites, during multiple
cycles of unwinding and translocation through a duplex region of DNA.
The dimeric Rep protein and the hexameric T7 gene 4 helicase have been
most rigorously studied and currently provide paradigms for an
understanding of helicase-catalyzed unwinding. The T7 gene 4 helicase
is thought to encircle the ssDNA molecule along which it translocates
and utilizes alternate protomers of the hexamer to unwind the duplex
region (46). The dimeric Rep protein has been proposed to unwind duplex
DNA via a mechanism in which the two protomers alternate binding to
ssDNA and dsDNA at the ssDNA/dsDNA junction during cycles of nucleotide
binding, hydrolysis, and product release (15). This mechanism is termed
the "rolling mechanism" since one can envision the helicase
subunits rolling through the duplex region.
Since Rep protein and UvrD share approximately 40% amino acid identity
and several biochemical properties, it has been suggested that the two
proteins are likely to unwind duplex DNA by the same mechanism
involving a dimeric helicase (20). However, we have presented
compelling evidence in this report to indicate that the monomeric form
of UvrD is an active helicase both in vitro and in
vivo. Therefore, an unwinding mechanism involving a monomeric helicase must be considered, and it is likely that Rep and UvrD unwind
DNA by substantially different mechanisms.
The assembly state of active UvrD has been a matter of speculation for
some time. Previous studies using gel filtration and protein-protein
cross-linking (19) provided evidence to suggest that UvrD forms dimers
and higher order oligomers. Protein-protein cross-linking can detect
very weak or transient interactions. However, interactions observed
with this technique need to be confirmed with other methods because
they may not reflect true association (47). Therefore, we used other
reliable physical techniques to investigate the assembly state of UvrD.
Gel filtration studies clearly suggest the existence of a population of
UvrD monomers and oligomers (presumably dimers) in equilibrium. In addition, analytical equilibrium ultracentrifugation studies using purified UvrD indicate that the enzyme can form dimers with a Kd for dimerization of 3.4 µM. This is
the first report of a dissociation constant for the dimerization of
UvrD. Interestingly, ATP and/or ssDNA ligands did not enhance the
dimerization of UvrD as has been reported for other helicases such as
Rep (13, 14) and the phage T7 gene 4 protein (8). In fact,
sedimentation velocity ultracentrifugation and gel filtration
chromatography experiments suggest that ATP and ssDNA stabilize the
monomeric form of UvrD. It is important to note that the dimerization
constant reported here is fairly high in relation to the concentration of UvrD present in the cell (0.3-0.8 µM (21)).
Therefore, oligomerization of the protein may not be relevant under
normal growth conditions (see below), although the possibility of
non-ideal behavior in vivo resulting in a lower apparent
dimerization constant cannot be rigorously excluded.
By using the yeast two-hybrid system we identified a UvrD mutant
lacking the C-terminal 40 amino acids that potentially possessed a
dimerization defect. Equilibrium ultracentrifugation and gel filtration
chromatography confirmed that purified UvrD
40C was not able to
dimerize in vitro in the absence of ligands. Similar to
results obtained with wild-type UvrD, ATP, and/or ssDNA did not induce
dimerization of UvrD
40C. However, this protein maintained full
biochemical activity as a helicase and a ssDNA-stimulated ATPase and
was fully functional in vivo in DNA repair. The simplest interpretation of these results is that UvrD
40C is an active monomeric helicase.
The biophysical data clearly indicate that UvrD
40C exists as a
monomer in solution and, whereas UvrD is capable of dimerization, it
too exists as a monomer at protein concentrations typically used for
in vitro enzymatic assays. Additional biochemical
experiments using wild-type UvrD yielded results that were also
consistent with a monomeric helicase. The ATPase and helicase
activities of UvrD were not significantly inhibited by the presence of
a molar excess of ATPase and helicase-deficient UvrD point mutants. Inhibition in these experiments would be expected if dimers or oligomers were required for activity but not if monomers were sufficient for activity. A previous study (21) reported inhibition of
UvrD-catalyzed unwinding by the UvrD-K35M mutant. This result was
likely due to limiting concentrations of DNA which resulted in
competition between wild-type and mutant monomers for the substrate molecules. Based on all the biophysical and biochemical data obtained in this study, we conclude that dimerization of UvrD (or UvrD
40C) is
not required for activity as a ssDNA-stimulated ATPase nor is it
required for helicase activity.
We also note that genetic studies shown here, and reported previously,
are consistent with the notion that UvrD is active as a monomer.
UvrD
40C was fully functional in vivo, and since the
helicase activity of UvrD has been shown to be required for activity in
mismatch and excision repair (21, 23, 24, 37), monomeric UvrD
40C
(and, by inference, monomeric UvrD) must be active as a helicase in
both repair pathways. In addition, a variety of uvrD alleles
that failed to function in these DNA repair pathways was recessive to
wild-type uvrD (21, 23, 45). The recessive nature of the
mutant uvrD alleles examined here suggests that the UvrD
helicase activity required in UV excision and methyl-directed mismatch
repair does not involve oligomerization. In similar studies, a dominant
phenotype has been demonstrated for mutants of the herpes simplex virus
type 1 UL9 helicase (48), a protein that is believed to function as a
dimer (49, 50).
Taken together, the studies reported here strongly support the
conclusion that the active species of UvrD is a monomer and that
oligomerization of the protein is either irrelevant or important only
when the cellular concentration of UvrD is very high (e.g. after SOS induction as discussed below). In fact, these results provide
the first direct evidence for a helicase that is able to catalyze
duplex nucleic acid unwinding as a monomer. Previous studies using the
HCV RNA helicase (51) and purified RecB protein (52) have suggested
these enzymes may be functional monomers. It is important to note that
the three helicase crystal structures available are all monomers
(53-55), although it was argued that the dimeric form of Rep helicase
may be resistant to attempts at crystallization. Thus, it is likely
that several monomeric helicases exist. Therefore, a mechanism for
unwinding by a monomeric helicase must be considered.
Unfortunately, there is not enough mechanistic data available for
helicase II to provide a detailed description of its unwinding mechanism. One possible model is based on multiple DNA-binding sites
within the monomeric enzyme. This would allow for continuous translocation of the helicase along ssDNA and through dsDNA since at
least one binding site would be in contact with the DNA lattice at all
times. Processive translocation, made possible by the multiple DNA-binding sites, would likely be driven by conformational changes in
the enzyme triggered by nucleotide binding and hydrolysis that are
coupled to the actual unwinding event. There is evidence both for
helicase II translocation along ssDNA (56, 57) and conformational changes associated with ligand binding (24, 58), lending support to
this type of model. In addition, based on the crystal structure of the
Rep helicase complexed to ssDNA (54) and site-directed mutagenesis
studies of helicase II (24, 41),2 it is likely that ssDNA
is contacted by several distinct regions of the enzyme. Such a model
would be fundamentally similar to the "inchworm" model originally
proposed for E. coli Rep helicase (59, 60). Although less
likely, an entirely different type of model is possible for a monomeric
helicase in which the helicase does not actively translocate along the
DNA molecule. In this model, the enzyme preferentially binds to a
ssDNA/dsDNA junction. After each unwinding event, a new junction is
made available for another helicase molecule to bind.
If we assume that UvrD translocates along ssDNA during an unwinding
reaction, as previous studies have indicated (56, 57) and as suggested
by the pseudo-single turnover helicase assays performed here, then a
version of the inchworm model for DNA unwinding may be a suitable
working model for helicase II-catalyzed unwinding (Fig.
7). This model assumes at least two
non-equivalent DNA-binding sites on the monomeric protein. The leading
site (L) must have an affinity for duplex DNA and may also
bind ssDNA, whereas the trailing DNA-binding site (T) need
only have an affinity for ssDNA. The binding of ATP, its subsequent
hydrolysis, and product release would cause the protein to cycle
between two or more conformational states as the protein "inches"
along the DNA. A cycle of unwinding begins with the enzyme in an
"extended" conformation (Fig. 7A), in which the
T site is bound to ssDNA and the L site is
extended forward in the vicinity of the ssDNA/dsDNA junction. Binding
of ATP to the enzyme triggers tight binding of the L site to
the ssDNA/dsDNA junction and induces a conformational change in the protein to a more compact state in which the T site is
shifted forward along the DNA lattice with respect to the L
site (Fig. 7B). This results in a transient high affinity
DNA binding state. In support of this idea sedimentation velocity
ultracentrifugation experiments reported here have shown that the
ATP-bound conformational state is more compact than the ATP-free state.
In addition, a previous report demonstrated that the ATP-bound state
possesses a higher affinity for DNA (24). Upon ATP hydrolysis, a
distinct number of base pairs (4-5 according to a recent report (20)) are disrupted at the ssDNA/dsDNA junction, and product release is
associated with a return of the enzyme to its original conformation by
extension of the L site forward with respect to the
T site (Fig. 7C). At this point, the L
site is in close proximity to the new ssDNA/dsDNA junction, and the
cycle is repeated to catalyze further unwinding.

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Fig. 7.
Model for duplex DNA unwinding by a monomeric
helicase. A hypothetical model based on available data was
generated to describe the mechanism of duplex DNA unwinding catalyzed
by the monomeric UvrD helicase. Details of the mechanism are described
in the text. The shapes of the helicase and the locations of DNA and
ATP-binding sites are arbitrary.
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