From the Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030
Received for publication, August 24, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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UL9, an essential gene for
herpes simplex virus type 1 (HSV-1) DNA replication, exhibits helicase
and origin DNA binding activities. It has been hypothesized that UL9
binds and unwinds the HSV-1 origin of replication, creating a
replication bubble and promoting the assembly of the viral replication
machinery; however, direct confirmation of this hypothesis has not been
possible. Based on the presence of conserved helicase motifs, UL9 has
been classified as a superfamily II helicase. Mutations in conserved
residues of the helicase motifs I Helicases, enzymes that unwind double-stranded nucleic acids (DNA,
RNA, or DNA/RNA hybrids), function in essentially every cellular
process involving nucleic acids (1, 2). Nucleic acid unwinding is a
dynamic process involving NTP binding/hydrolysis, hydrogen bond
breakage, and translocation of the helicase along single-stranded
nucleic acid, accompanied by conformational changes in the protein
molecule itself (1-3). Despite their diversity with respect to
sequence, size, oligomeric state, and substrate preference, all
helicases possess conserved motifs proposed to play pivotal roles in
helicase activity (4). Two superfamilies of helicases,
SFI1 and SFII, have been
characterized by the presence of seven conserved motifs. Between these
two superfamilies, motifs I and II are well conserved; however, motifs
Ia and III-VI exhibit very limited homology2 (3). Despite this
limited sequence similarity, crystallographic information shows
remarkable structural similarity between helicases from SFI and SFII
(4-7). The structures of helicases from both superfamilies (Rep from
Escherichia coli (2), PcrA from Bacillus stearothermophilus (1), NS3 helicase from hepatitis C virus (8-10), UvrB from Bacillus caldotenax (11) and
Thermus thermophilus (12), and eIF4a from
Saccharomyces cerevisiae (13)) reveal the presence of two
RecA-like domains. The conserved helicase motifs are positioned along a
cleft that runs between the two Rec A-like domains comprising an
ATP-binding site. The adenine moiety of ATP is positioned at the base
of the ATP-binding cleft, and phosphates protrude upward inside the
cleft. ATP binding in the cleft results in conformational changes
leading to cleft closure (8, 14, 15). Many helicases require a divalent
metal ion, preferably Mg2+, that is believed to play an
important role in the correct positioning of the ATP phosphates in the
binding site as well as for the destabilization of the P-O bond,
facilitating hydrolysis (16). A conserved lysine from the GS/TGKT
(motif I) was shown in the crystal structures of several helicases to
coordinate ATP The mechanism of helicase action is believed to involve the coupling
between several steps including ATP binding, ATP hydrolysis, and
binding to nucleic acid. The Rep, PcrA, and NS3 helicase structures have also been solved in complexes with nucleic acids as follows: Rep
with ssDNA (2), PcrA with partially dsDNA (18), and the NS3 helicase
with ssRNA (8). In all three structures, single-stranded nucleic acid
was observed to bind in a cleft almost perpendicular to the ATP-binding
site, positioned at the top of the RecA-like domains. Structural and
genetic data suggest that residues from motifs Ia, III, and V are
involved in ssDNA/RNA binding (2, 8, 18). Motifs III-VI have been
implicated in the coupling between the ATPase and helicase activities
and/or in the coupling between ATPase and ssDNA binding activities (8,
19-22).
The UL9 gene is essential for herpes simplex virus type 1 (HSV-1) replication in vivo (23) and exhibits helicase and
origin binding activities in vitro (24, 25). UL9 is composed
of at least two domains (Fig. 1) as follows: a C-terminal
origin-binding domain (residues 535-851) and an N-terminal helicase
domain (residues 1-535) that contains the SFII-conserved helicase
motifs (3). Each domain expressed separately exhibits the expected
biochemical activities (26, 27). UL9 has been hypothesized to bind and unwind the HSV-1 origin of replication, creating a replication bubble
and promoting the assembly of the viral replication machinery (28-30);
however, direct confirmation of this hypothesis has been lacking. The
importance of UL9 in HSV-1 replication in vivo has been
supported by genetic experiments (23, 31). Studies with temperature-sensitive mutants demonstrated the importance of
UL9 during the early stages of viral infection (31). The
overexpression of wild type UL9 or the origin-binding domain
by itself is inhibitory to HSV-1 replication (32-35). The mechanism of
HSV-1 origin unwinding is poorly understood, and it has not been
possible to demonstrate the unwinding of duplex origin-containing
plasmids in vitro. Previous attempts to unwind
oriS-containing helicase substrates in vitro have revealed
the requirement for the presence of ssDNA near the origin; moreover,
unwinding could be observed only in the presence of HSV-1 ssDNA-binding
protein, ICP8 (36, 37). Electron microscopic studies have demonstrated
that unwound stem-loop structures form in the presence of UL9 and HSV-1
ssDNA-binding protein (38). Due to the artificial nature of the
substrates (36, 37) and the inability to demonstrate unwinding of
duplex origin in vitro, the significance of the helicase
function of UL9 for HSV-1 replication remains to be determined.
A collection of mutations in conserved residues of UL9
helicase motifs I-VI have been isolated, and most fail to complement the growth of UL9 null virus in vivo (39). In
this paper, we report that mutations in motifs I-IV and VI exhibit
decreased levels of ATPase activity and most abolish helicase activity, but they do not interfere with UL9 dimerization or the ability of UL9
to bind the HSV-1 origin of replication. Based on the similarity of the
helicase motif sequences between UL9 and UvrB, a superfamily II helicase-like enzyme, we were able to map the UL9
helicase motif mutations on the structure of UvrB and provide an
explanation for the observed mutant phenotypes (40). Our results
indicate that the helicase function of UL9 is indispensable for viral
replication in vivo, supporting the hypothesis that UL9 is
essential for unwinding at the HSV-1 origin of replication.
Furthermore, the data presented provide insights into the mechanism of
transdominance of the UL9 helicase motif mutants.
Reagents and Materials--
Supplemented Grace's medium and
penicillin/streptomycin were purchased from Life Technologies, Inc.;
fetal calf serum was from Gemini Biological Products. Restriction
enzymes were from New England Biolabs, and protease inhibitors and
ampicillin were from Sigma. BaculoGoldTM DNA was purchased
from PharMingen, and the pFastBac vector and DH10BacTM
competent cells were from Life Technologies, Inc. The SP-Sepharose and
Superose 12 HR 10/30 columns were from Amersham Pharmacia Biotech, and
the UnoS column was from Bio-Rad.
Cells--
Spodoptera frugiperda (Sf9) insect
cells were maintained in Grace's medium supplemented with 10% (v/v)
fetal calf serum, 0.1 mg/ml streptomycin, and 100 units/ml penicillin.
E. coli DH5 Western Blot Analysis--
Western blot analysis was performed
as described previously (41). In brief, samples were resolved by
SDS-PAGE and electrotransferred to HybondTM
ECLTM membrane using Tris/glycine/methanol transfer buffer.
Membranes were blocked with 5% milk in Tris-buffered saline, incubated
with anti-UL9 primary antibody and alkaline phosphatase-conjugated secondary antibody (Promega), and visualized by the alkaline
phosphatase color reaction. Three different primary antibodies were
used as follows: 1) the R250 polyclonal antibody, recognizing the
C-terminal residues 841-851, was generously provided by Dr. M. Challberg (National Institutes of Health, Bethesda); 2) RH7, a
polyclonal antibody raised against the C-terminal domain of UL9, was
generously provided by Dr. Daniel Tenney (Bristol-Myers Squibb Co.);
and 3) 17B, a monoclonal antibody, recognizing the N-terminal 35 amino acids of UL9, was described previously (42).
Generation of Recombinant Baculoviruses Expressing
UL9--
Mutations in the helicase motifs of UL9 were
described previously: motif I (UL9-K87A), motif II (UL9-E175A), motif
III (UL9-T214S), motif IV (UL9-F303W), motif V (UL9-G354A), and motif
VI (UL9-R387K) (39). Hereafter we will refer to the UL9
helicase motif mutants only by the relevant motif number. The motif
mutations were initially constructed in p6-119b, a vector containing
the ICP6 promoter (39). The wild type UL9 gene was subcloned
into a baculovirus transfer vector (pP10 or pFastBac) as a
BamHI fragment. Each of the UL9 mutant genes was
cloned into the baculovirus vector by ligation of a 1.1-kilobase pair
NheI-EcoNI fragment from the corresponding p6UL9-119b plasmids (39) to NheI/EcoNI-digested
pP10-UL9 or pFastBac-UL9, respectively.
Recombinant baculoviruses (Autographa californica nuclear
polyhedrosis baculoviruses) expressing wild type or mutant UL9
protein were generated using the baculovirus transfer vectors,
described above, and the BaculoGold (PharMingen) or pFastBac (Life
Technologies, Inc.) commercial systems according to manufacturers'
instructions. The recombinant baculoviruses were amplified to produce
large scale baculoviral stocks, tested for optimal expression in
Sf9 insect cells, and used further for protein production (see below).
UL9 Expression and Purification--
Sf9 cells in 50%
confluent flasks were infected with the recombinant baculovirus of
interest, and the cells were harvested at 48 h post-infection by
vigorous shaking. The cell pellet was washed once with
phosphate-buffered saline, resuspended in Buffer B (20 mM
HEPES, pH 7.6, 1 mM EDTA), and homogenized in a Dounce homogenizer. The nuclei were pelleted in a GSA rotor at 3,000 rpm for
10 min at 4 °C. The nuclear pellet was resuspended in 30 ml of
Buffer B with 10% sucrose and mixed with an equal volume of 3.4 M NaCl in Buffer B. High speed centrifugation (27,000 rpm, SW 28 rotor, Beckman ultracentrifuge) was used to separate the soluble
and insoluble protein fractions. The soluble proteins were precipitated
with ammonium sulfate. The majority of UL9 was found to precipitate at
50% saturation. The ammonium sulfate precipitate was resuspended in a
minimal volume of Buffer B containing 0.25 M NaCl and
dialyzed overnight against 4 liters of the same buffer. The dialyzed
sample was centrifuged to remove insoluble particles and subjected to
chromatography on an SP-Sepharose column. The protein bound to the
column was eluted with a 0.25-1 M linear gradient of NaCl.
UL9 was found to elute reproducibly at 0.45-0.6 M NaCl.
SP-Sepharose fractions were monitored by the Bradford protein assay and
Western blot with anti-UL9 antibodies. Fractions containing UL9 were
pooled and loaded on a UnoS column. Protein elution and fraction
screening were performed as described above for the SP-Sepharose
column. UnoS fractions containing UL9 were concentrated using a
MicrosepTM concentrator (Pall Filtron Corporation) with a
30-kDa cutoff and loaded on a Superose HR 10/30 gel filtration column.
The fractions containing UL9 were identified and concentrated as
described above. Aliquots of purified protein in buffer B containing
0.25 M NaCl were stored at Protein Microsequencing--
Protein microsequencing was
performed to identify a contaminating protein with a molecular
mass of ~40 kDa seen in Coomassie-stained SDS gels of purified
wild type and mutant UL9. Five micrograms of the wild type UL9
preparation was resolved on 10% SDS gel and electrotransferred to
Immobilon-PSQ membrane (Millipore; pore size 0.2 µm) using the
CAPS/methanol buffer system. The membrane was stained with Coomassie
and the band of interest excised. Membrane elution and protein
microsequencing were performed in the protein sequencing facility of
University of Massachusetts Medical School, Shrewsbury, MA, by Dr. J. Leszyk.
Limited Proteolysis--
Protease inhibitors used during the
protein purification scheme were removed by chromatography on a
Sephadex G-75 gel filtration column using 10 mM Tris-HCl,
pH 8.0, 0.1 M NaCl, 1 mM EDTA, 1 mM
DTT buffer and flow rate of 1 ml/min. Fractions containing UL9 were
identified with the Bradford protein assay and used for proteolysis.
The proteolytic reactions were performed in 10 mM Tris-HCl,
pH 8.0, 0.2 M NaCl, 1 mM EDTA, 1 mM
DTT, 10 mM CaCl2 buffer (43). Each reaction (1 ml) contained 30 µg of UL9 protein and 1:100 (v/v) 1 mg/ml solution
of proteinase K. The proteolysis was performed on ice. Aliquots of 330 µl were removed at 15, 30, and 60 min. The reaction was stopped by
adding phenylmethylsulfonyl fluoride to a final concentration of 10 mM, and the reaction mixtures were vortexed and frozen on
dry ice. After completion of the time course all samples were thawed,
and trichloroacetic acid was added to 10% (v/v) final concentration,
and protein precipitation was carried out on ice for 30 min. The
samples were spun down, washed with ice-cold ethanol, air-dried, and
dissolved in SDS-PAGE sample buffer (200 mM Tris-HCl, pH
8.8, 100 mM DTT, 2% SDS, 10% glycerol). The samples were
resolved on 10% acrylamide gels, transferred to an ECL nitrocellulose
membrane, and analyzed by Western blot with anti-UL9 antibodies. Life
Technologies, Inc., prestained molecular weight markers were used as a
reference for protein size.
ATPase Assay--
ATPase activity was monitored by the Malachite
Green/ammonium molybdate colorimetric assay as described previously
(24, 25, 44). M13 ssDNA was prepared as described previously (45) and
used as a DNA effector at 20 µM final concentration in
nucleotides, unless otherwise stated.
Helicase Assay--
The helicase substrate was generated by
annealing M13 ssDNA and a 45-nucleotide-long
5'-32P-end-labeled (46), resulting in a partially
double-stranded substrate with a 23-nucleotide 5' ssDNA
overhang. The substrate was annealed in buffer containing 6 mM Tris-HCl, pH 7.5, 7 mM MgCl2,
0.1 M NaCl, and 1 mM DTT. The annealing
reaction was heated to 70 °C for 10 min and slowly cooled to room
temperature. The annealed substrate was incubated for 30 min at
37 °C in the presence of 1 mM EDTA and purified twice on
Sephacryl 200 column to separate the annealed substrate from the free.
The helicase assay was performed in HEPES-based buffer (20 mM HEPES, pH 7.6, 1 mM DTT, 5 mM
MgCl2, 10% glycerol, 0.5 mg/ml BSA, 0.2 M
NaCl) for 30 min at 37 °C. Each helicase reaction (50 µl)
contained 1 nM helicase substrate and 200 nM
UL9 protein (unless otherwise stated). The reaction was stopped with
the addition of 25 µl buffer containing 0.1 M EDTA, 40%
glycerol, 0.1% bromphenol blue, resolved on 8% native polyacrylamide
gels, and visualized by autoradiography. Helicase reactions boiled for
10 min before loading were used as a reference for the mobility of the
unwound, and the helicase reaction without UL9 protein was used as a
reference for the mobility of the annealed substrate.
Filter Binding Assay--
A double filter nitrocellulose filter
binding assay was performed to evaluate the ability of wild type and
mutant UL9 proteins to bind the HSV-1 origin of replication. Each
reaction contained 1× filter binding buffer (50 mM HEPES,
pH 7.6, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10% glycerol), 0.2 mM NaCl, 0.4 nM DNA substrate (unless otherwise stated), 100× (w/w)
excess of poly(dI-dC), and 5 mg/ml BSA. A 10-well filter
apparatus (Hoefer) was used to address the specificity of origin
binding. The DNA substrate for the filter binding assay was prepared as
follows: the p-100-1 plasmid, containing OriS, was digested with
MspI, and the resulting single-stranded overhangs were
filled in with Klenow polymerase reaction in the presence of
[32P]dCTP under standard conditions (45). Circles of
nitrocellulose (BA85; Schleicher & Schuell, pore size 0.45 µm) and
DEAE-cellulose (NA 45, Schleicher & Schuell, pore size 0.45 µm)
membranes were prepared as described previously (47). Filter binding
reactions were filtered through the double membrane. In this assay the
nitrocellulose membrane is expected to retain protein-DNA
complexes, and DEAE-cellulose membrane is expected to retain the
unbound DNA. The nonspecifically bound DNA was eliminated from the
nitrocellulose membrane by washing with 1 ml of filter binding buffer.
The filters were collected, and the bound DNA was eluted as follows.
Nitrocellulose membranes were incubated for 2 h at room
temperature with 1 ml of 1% SDS, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA buffer, and the eluted DNA was precipitated with
ice-cold ethanol. The same procedure was used to elute the DNA bound to
the DEAE-cellulose membrane except the elution buffer was supplemented
with 1 M NaCl. The precipitated DNA was dissolved in water,
and aliquots were counted in a Beckman LS2800 scintillation counter.
The eluted DNAs were resolved on native acrylamide gels and visualized
by autoradiography. The ability of wild type and mutant UL9 to bind the
origin of replication was monitored by a dot-blot filter binding assay
(47), also using nitrocellulose and DEAE-cellulose membranes. A
gel-purified MspI fragment of p-100-1 plasmid containing
OriS was labeled as described above and used as DNA substrate. After
filtration, the nitrocellulose and DEAE-cellulose membranes were
air-dried, and DNA retention was quantitated with a PhosphorImager.
Generation of Recombinant Baculoviruses and Purification of Wild
Type and Mutant UL9 Proteins--
The baculovirus expression system
was shown to produce functional wild type UL9 protein exhibiting
ATPase, helicase, and origin binding activities (24, 25). To evaluate
the importance of conserved residues in the UL9 helicase motifs, we
constructed recombinant baculoviruses expressing wild type and mutant
versions of the UL9 protein (Fig. 1). All
recombinant baculoviruses except the motif V mutant expressed intact
full-length protein as detected by Western blot analysis using
antibodies against the N terminus (17B) and the C terminus (R250) of
UL9 (data not shown). Wild type and mutant UL9 proteins were purified
to greater than 80% homogeneity (Fig. 2)
as seen by Coomassie-stained SDS-PAGE. A contaminating band with a
molecular mass of ~40 kDa was observed in all preparations.
Microsequencing of the first five N-terminal amino acids revealed that
the contaminating protein is most likely an early 39-kDa protein
(Swiss-Prot accession number P11042) from A. californica
nuclear polyhedrosis virus, the baculovirus used for protein
expression. It is known that this gene is required for very late gene
expression and that the protein is associated with the nuclear matrix
(48). Our results (shown below) indicate that the contaminant did not
interfere with the biochemical assays performed in this study. For
example, a comparison of the kinetic parameters for wild type ATPase
activity with already published values and the broad spectrum of
effects on ATPase activity seen in various mutant UL9 preparations show
that the early 39-kDa protein does not influence the ATPase assay. In
addition, the absence of helicase activity in most mutant preparations
indicates that the contaminant protein does not exhibit helicase
activity.
All purified proteins except the motif IV mutant behaved similarly
during the course of purification, eluting from the ion-exchange columns as a single peak (data not shown). In contrast, the motif IV
mutant did not elute as a discrete peak but was found in the majority
of the fractions eluted from the ion-exchange columns, independent of
the salt concentration of the eluting gradient. Such behavior suggests
that the mutation may alter the overall distribution of surface charges
on the molecule and consequently its ability to bind to the
ion-exchange column. Indeed, in gel filtration experiments, the motif
IV mutant protein was found to aggregate (see below). Our attempts to
express the motif V mutant protein, previously shown to be unstable in
mammalian cells (40), did not yield any stable full-length protein;
therefore, it was impossible to purify and characterize biochemically
this mutant.
All Helicase Motif Mutants Except the Motif IV Mutant Are Able to
Dimerize--
It has been reported previously that wild type UL9 is a
dimer in solution (24, 25), whereas the C-terminal domain by itself is
a monomer (49). These results imply that the UL9 residues responsible
for dimerization are localized in the N-terminal two-thirds of the
protein. Gel filtration chromatography was performed as a step in the
purification of UL9 as well as to determine the oligomeric state of UL9
helicase motif mutants. Wild type UL9 was found to behave as reported
previously (24), eluting from the Superose HR 10/30 gel filtration
column as a wide peak centered around a position corresponding to a
molecular mass of 180 kDa (Fig. 3). All
motif mutants except the motif IV mutant showed a similar elution
pattern (Fig. 3). The motif IV mutant was shown to aggregate, eluting
as a discrete peak corresponding to a molecular mass above 443 kDa.
Since the gel filtration column used is linear only up to 300 kDa
(Amersham Pharmacia Biotech), it was not possible to determine
precisely the molecular weight of the aggregates. Aggregates were also
observed in the preparation of the motif VI mutant, but in this case
most of the mutant protein eluted as a dimer. These results argue that
the helicase motifs are most likely not involved in UL9 dimerization.
This result provides support for the idea that residues outside the
helicase motifs are involved in protein-protein interactions (2).
Mutations in the Helicase Motifs Do Not Drastically Affect the
Overall Conformation of the UL9 Molecule--
Limited proteolysis
using proteinase K was performed to evaluate the effect of the
introduced mutations on the overall conformation of the UL9 protein.
Since the yield of the UL9 protein is a limiting factor for extensive
proteolytic studies, we took advantage of the availability of anti-UL9
antibodies recognizing the N terminus (17B) and the extreme C terminus
(R250) of UL9. Western blot analysis was used to follow the fate of the
proteolytic fragments containing each of the termini. Proteinase K
digestion produced two major C-terminal fragments (Fig.
4A, asterisks) and one major
N-terminal fragment (Fig. 4B). All UL9 mutants, except the
motif VI mutant, showed the same general proteolytic pattern as wild
type suggesting that the mutations introduced did not significantly
alter the overall conformation of the protein. Motif VI digestion did
not generate a detectable N-terminal fragment (Fig. 4B, lanes
24-26) suggesting that the mutation induces local conformational
changes in the UL9 molecule. These changes most probably affect the
accessibility of the extreme N terminus, where the antibody epitope
maps, since the Western blot analysis with the anti-C-terminal antibody
showed that a C-terminal fragment slightly shorter than the full-length UL9 was present throughout the entire time course (Fig. 4A, lanes 24-26). Some differences in the kinetics of the proteolysis were evident (Fig. 4A, compare lanes 1-4 (wild type
UL9) with lanes 19-22 (motif IV) and Fig. 4B,
compare lanes 1-4 (wild type UL9) with lanes
19-22 (motif IV) and lanes 23-26 (motif VI)). Motif IV produced less stable C-terminal fragments that were almost completely degraded by 60 min. The motif VI mutant protein was degraded
slightly faster than the wild type UL9 protein (compare Fig. 4B,
lanes 1-4 with lanes 23-26). Additional bands with
low abundance were seen in the Western blot with R250 antibody (Fig. 4A). They may be intermediate products of proteolysis. These
results suggest that the helicase motif mutations do not drastically
alter the overall conformation of UL9, but minor conformational changes have not been excluded. Surprisingly, no drastic changes in the proteolytic pattern were observed even for the motif IV mutant, shown
to aggregate (see above and Fig. 3). This finding suggests that the
aggregates are composed of folded molecules with minor changes in
conformation rather than of completely unfolded polypeptide chains. In
the latter case we would expect to see no stable proteolytic fragments.
The proteolysis experiment suggests that the phenotypes observed
(described below) reflect a direct effect of the introduced mutation on
UL9 function rather than an indirect effect due to global changes in
conformation. The phenotypes of the motif IV and motif VI mutants
should be interpreted with caution due to the possible local changes in
the conformation of the corresponding proteins as seen by
proteolysis.
Mutations in All Helicase Motifs Affect the Ability of UL9 to
Bind/Hydrolyze ATP--
The ATPase activity of wild type and mutant
UL9 proteins was examined using the Malachite Green/ammonium molybdate
colorimetric assay (44). Time courses of intrinsic (Fig.
5A) and ssDNA-stimulated (Fig.
5B) ATPase activity were performed to determine the relevant rate constants (Fig. 5C). Wild type UL9 was found to
hydrolyze ATP with a rate constant of 2.96 min The Helicase Activity of UL9 Was Abolished by Mutations in All
Helicase Motifs Except Motif III--
The effect of the helicase motif
mutations on the helicase activity of UL9 was evaluated using an
in vitro unwinding assay. In the absence of ATP, no
unwinding was observed (data not shown). When ATP was supplied to the
reaction, only wild type and the motif III mutant exhibited helicase
activity (Fig. 6A). To compare the helicase activities of these two proteins, we performed unwinding reactions varying the amount of UL9 protein added. We found that the
amount of unwound DNA correlated with the amount of UL9 present in the
reaction and that the helicase activity of the motif III mutant was
comparable to that of wild type (Fig. 6B). PhosphorImager quantitation indicated that when UL9 (wild type or the motif III mutant) was present at a concentration of 200 nM, 60% of
the helicase substrate is unwound. When UL9 was present at 100 or 50 nM, the percentages of unwound substrate were 35 and 20%,
respectively. Thus, the mutations in all conserved helicase motifs of
UL9, except motif III, abolished the helicase function of
the UL9 protein; the motif III mutant protein exhibited helicase
activity comparable to that of wild type UL9, which may not be
surprising taking into account the conservative nature of the
substitution of serine with threonine.
The phenotype of the motif III mutant is intriguing. It exhibited wild
type levels of intrinsic ATPase and helicase activity, and a moderate
defect in ssDNA-stimulated ATPase activity. Despite possessing wild
type levels of in vitro helicase activity, it complemented
the growth of hr94, the UL9 null virus, in vivo
only partially (39). One possible explanation for this apparent
discrepancy may reflect the different nature of the assays, in
vitro versus in vivo. Alternatively, the
ssDNA stimulated ATPase activity may play a role in addition to its
proposed role in the helicase function of UL9. Another possibility is
that the mutation may alter crucial protein-protein or protein-DNA
interactions essential for replication but not measured by the assays
used. Any of these effects may explain the partial complementation phenotype.
Mutations in the Helicase Motifs of UL9 Do Not Alter Its
Origin-specific DNA Binding Activity--
To determine the effect of
the helicase motif mutants on the origin-binding function of UL9, a
double membrane filter binding assay was performed. Our analysis
indicated that none of the characterized mutations in the helicase
motifs of UL9 interferes with the specificity of the origin binding
(Fig. 7). When MspI-digested
p-100-1 plasmid (Fig. 7, lane 15) was used as a substrate in
the filter binding reaction, only the OriS-containing fragment was
selectively retained on the nitrocellulose membrane by the wild type
and all motif mutant proteins (Fig. 7, lanes 8-14) even
though multiple labeled plasmid fragments with similar length were
present.
To quantify the origin binding abilities of wild type and mutant
proteins, we chose to compare OriS binding of wild type and mutant UL9
at protein concentrations (1, 4, and 10 nM), shown to be in
the linear range of the binding curve of UL9 to an OriS-containing DNA
fragment (24). Our experiments indicated that all characterized helicase motif mutants, except motif IV mutant, bind OriS with wild
type efficiency at all concentrations examined (Fig.
8). The binding of the wild type UL9
protein was plotted as 100%. At 1 nM, 5% of the DNA
substrate in the reaction was bound by wild type UL9. At 4 and 10 nM, the percentages of bound substrate were 12 and 30, respectively. The differences observed between wild type and mutant UL9
proteins, with the exception of the motif IV mutant protein, were less
than 2-fold. The motif IV mutant exhibited a 4-fold lower than wild
type binding efficiency to OriS at the 10 nM protein
concentration. This result may reflect the tendency of the mutant
protein to aggregate, which is not predominant when the protein is at
lower concentrations. In conclusion, all UL9 motif mutant proteins,
except motif IV, exhibited near wild type ability to bind OriS.
Previous genetic studies of UL9 helicase motif mutants
showed that the conserved residues are important for UL9
function in vivo since none of the mutants were able to
complement fully the growth of the UL9 null mutant (hr94).
In fact, partial complementation was observed only for the motif III
(UL9-T214S) mutation, which we now show retains helicase activity (Fig.
6). However, when the same position was mutated to alanine, the UL9
function was abolished completely (39). The biochemical analysis
reported in this study correlates very well with the genetically
observed phenotypes of the helicase motif mutants (39). Thus, the most likely reason for the inability of mutants in motifs I-IV and VI to
complement the growth of the UL9 null virus is that helicase activity is completely abolished. Although direct evidence for UL9
unwinding of the origin of replication in vivo is still
lacking, the most straightforward explanation for our results is that
UL9 plays an indispensable role in HSV-1 origin unwinding.
Functions of the Conserved Helicase Motifs--
The UL9
helicase motif mutants were designed based on the conservation of the
targeted residues within helicase motifs of UL9 homologs
(39) before any structural information about helicases was available.
Sequence alignment (Fig. 9A)
of the helicase motifs of UL9 with the helicase motifs of
UvrB allowed us to map the mutated residues in UL9 on the
structure of UvrB (Fig. 9B) and gain additional insights
into the molecular basis behind the observed mutant phenotypes. UvrB is
a member of nucleotide excision repair pathway, which exhibits limited
helicase activity only when complexed with UvrA (51, 52). Intriguingly,
like UL9, UvrB is not able to separate long DNA duplexes (52). Thus
neither UvrB nor UL9 when expressed alone exhibit very robust helicase
activity on natural substrates. It is tempting to speculate that
similarities in the biochemical properties of UL9 and UvrB may be due
to their structural similarities.
ATP Binding and Hydrolysis--
Motifs I and II (Walker box A and
B, respectively) were shown previously to play an important role in ATP
binding/hydrolysis by mutational analysis and crystallographic studies
(reviewed in Refs. 4, 6, and 53). The motif I Lys-87 residue of UL9
corresponds to Lys-45 in UvrB, seen in the crystal structure to contact
the
The glutamate at position 175 from motif II (Walker box B) of UL9
corresponds to Glu-339 from UvrB, seen to be oriented toward the ATP
molecule bound but at distance too great to be involved in direct
interaction (11). A glutamate in Walker box B of two other ATP-binding
proteins (Rec A (17) and PcrA (14)) was hypothesized to be responsible
for the activation of a water molecule involved in attacking the P-O
bond. Indeed, in PcrA, when this glutamate was replaced with an
alanine, a 70-fold decrease of the kcat for
ssDNA-stimulated ATPase activity and no significant effect on
Km for ATP were observed. In UL9 the same type of
mutation resulted in almost undetectable intrinsic ATPase and a 6-fold
reduction of the ssDNA-stimulated ATPase activity. Surprisingly, the
ssDNA stimulation of the ATPase activity was 4-fold higher than that
for wild type (Table I), which correlates with a 5-fold lower
Km for ssDNA. A phenotype similar to that of the motif II mutant has been observed previously for the E221Q mutation in
UvrD (54). There is no straightforward explanation for the increased
ssDNA stimulation, and the lower Km value for ssDNA
since motif II has not been observed in any helicase/nucleic acid
structure in close proximity to the ssDNA-binding cleft. It is possible
that the mutant protein undergoes a conformational change upon ssDNA
binding and/or it is involved in hydrogen bonds or electrostatic
interactions with other residues involved directly or indirectly in
ssDNA binding. Alternatively, the inability of the mutant to hydrolyze
ATP could lead to arrest of the protein molecule in an ATP/ssDNA-bound
state. Thus, each mutant protein molecule may spend a longer time on
ssDNA due to the slower rate of release from the complex, which is seen
as an indirect effect on the Km for ssDNA. In
summary, our results confirm the importance of motifs I and II in ATP
binding/hydrolysis and indicate that motif II may also mediate
allosteric effects of ssDNA on ATPase activity.
Coupling of the ssDNA-binding ATPase and Helicase
Activities--
In addition to ATP binding and hydrolysis, multiple
events have to take place for helicase unwinding to proceed. ssDNA
binding activity, unwinding, and translocation along the unwound DNA
are tightly interrelated and coordinated with ATP binding/hydrolysis. Limited information is available about the specific mechanism of action
of any helicase, but mutational studies have suggested that motifs
III-VI may play a role in the coupling of ATP binding/hydrolysis to
ssDNA binding and helicase activities (7, 15, 19-21, 53, 55).
Motif III is positioned along the ATP-binding cleft (Fig.
9B) and can be pictured also as a bridge between the
ATP-binding and ssDNA-binding clefts suggesting a role in coupling the
ATPase activity with DNA binding and/or helicase activity. The
phenotype of the D248N mutant of motif III from E. coli
helicase II (UvrD) supports the notion that motif III is at the
crossroads between the ATPase and ssDNA binding activities. This mutant
was found to be defective in the formation of binary complexes with ATP and DNA separately but formed a wild type tertiary complex
(UvrD-ATP-ssDNA) as seen from the corresponding ATP/ssDNA binding
studies (20).
We report herein that UL9 motif III mutant behaves as wild
type in all activities except ssDNA-stimulated ATPase. The
Km values for ATP and ssDNA are comparable with the
wild type (Table I). In the crystal structure of UvrB, the
corresponding Thr-395 is oriented toward the ATP-binding pocket but too
far to be involved in direct contacts with ATP (11). The UL9 Thr-214
could be involved in hydrogen bonding with residues directly involved
in ATP and/or ssDNA binding. The slight change in the nature of the
side chain (threonine to serine) may result in a very mild effect of
uncoupling between the ATPase and ssDNA binding abilities reflected in
the decreased ssDNA-stimulated ATPase activity. A mutant with a
substitution of the threonine to alanine does not complement the growth
of hr94 virus, suggesting a functional/structural role for the hydroxyl group of Thr-214. Isolation and characterization of additional mutants
will be required to resolve the role of motif III more precisely.
Motif IV of SFII helicases has not been well studied genetically.
Mutations in conserved residues of motif IV of SFI helicases resulted
in defects in ATP binding and/or or uncoupling between the ATPase and
helicase activities (14, 19, 56). In the crystal structure of NS3
helicase bound to ssRNA, residues from motif IV were seen to contact
RNA via water molecules or backbone contacts (8). Calculations of the
surface electrostatic potential of the UvrB molecule showed that
residues from motif IV are involved in a positively charged surface
that potentially can bind DNA (11), but this has not been confirmed
experimentally. Taken together these observations imply that motif IV
in SFII may be involved in single-stranded nucleic acid binding, rather
than in the coupling between the ATPase and helicase activities. It is
possible that the roles of motif IV in SFI and SFII helicases are
completely different since these motifs differ in their position with
respect to the ATP and ssDNA-binding clefts. In SFI, motif IV is
positioned along the ATP-binding cleft spanning the two RecA-like
domains (1, 2), whereas in SFII it is positioned on top of one of the
RecA-like domains along the RNA-binding cleft at some distance from the
ATP-binding cleft (8-13).
In motif IV of UL9, the replacement of Phe-303 with tryptophan resulted
in an aggregated protein that still retains some ATPase activity and
somewhat impaired wild type levels of origin-specific DNA binding
ability. In UvrB, a threonine (Thr-452) instead of a phenylalanine is
present in this position (Fig. 9). Thr-452 is packed against residues
of several loops and is almost completely buried. Assuming that UL9
Phe-303 is also buried, it is possible that the presence of the bulkier
tryptophan results in local conformational changes and exposure on the
surface of residues, which are not normally exposed, leading to
aggregation. At this time we cannot differentiate between a direct
effect of the mutation on UL9 properties and an indirect effect of aggregation.
Motif VI of SFII helicases was proposed to be a gatekeeper of the
ATP-binding cleft (8) and to be involved in the coupling between the
ATPase and helicase activities (57), which has been confirmed by
mutagenesis (21, 58). A very striking case of uncoupling is seen in the
R544K mutation in UvrB (57). This mutant exhibits 3-fold higher
intrinsic ATPase activity than wild type but no helicase-like activity.
Similar phenotypes of uncoupling are seen for helicases from SFI UL5/52
(19), PcrA (14), and UvrD (55). In UL9, the replacement of Arg-387 with
lysine results in reduction of the intrinsic and ssDNA-stimulated
ATPase activities and complete abolition of the helicase activity. This
phenotype is consistent with the role of motif VI as a gatekeeper. The
analogous arginine in UvrB is located along the ATP-binding cleft above the ATP molecule (Fig. 9). It is possible that upon DNA binding, this
residue undergoes a conformational change and moves closer to ATP,
which may explain the observed phenotype.
Our results and biochemical characterization of helicase motif mutants
from other systems indicate that helicases are very complex enzymes.
Their mechanism of action cannot be understood by simple assignment of
function(s) to individual conserved helicase motifs. Helicase
complexity is demonstrated by studies in which multiple residues within
the same helicase motif are mutagenized. For example, when Trp-259 from
PcrA motif III was mutated to alanine, a significant effect on ssDNA
binding was observed, but all ATP binding/hydrolysis kinetic parameters
were comparable with wild type (22). In contrast, when Gln-54 from the
same motif was mutated, no effect on the ssDNA binding was observed,
but the ATPase activity was decreased (22).
In all SFI and SFII helicases studied to date, mutations in helicase
motifs I-VI exhibit defects in ATPase activity that can be explained
at least in part by the nature of the ATP-binding cleft, whose
architecture is shaped by residues of the helicase motifs. It appears
that the helicase molecule (in general) is a very dynamic structure.
Kinetic studies of Rep (59, 60), UvrD (20), and NS3 helicase (61, 62)
predict that the ATP and the ssDNA binding are multistep processes
accompanied by conformational changes. In UvrD and Rep these
conformational changes have been confirmed by limited proteolysis (20,
63).
The Mechanism of Transdominance of UL9 Helicase Motif
Mutants--
The study of transdominant mutants has provided valuable
information about protein functional domains and has facilitated the
understanding of the molecular mechanisms underlying many biological
processes. The finding that the N-terminal domain of UL9 expressed by
itself is a functional helicase (26, 64) and the C-terminal domain is a
monomer capable of binding the origin of replication (49) creates an
opportunity for distinguishing the roles of these two domains in HSV-1
infection. Previously, in a plaque reduction (transdominance) assay,
wild type UL9, when overexpressed, was found to be
inhibitory; mutants in motifs I, II, and VI were transdominant; mutants
in motifs III and IV were neutral, and the mutant in motif V was
potentiating (Table II and Ref. 40).
Several other lines of evidence also indicate that overexpression of
wild type UL9 is inhibitory. Malik et al. (65)
reported that although cell lines containing a low copy number of the
wild type UL9 gene could efficiently complement hr94, a
UL9 null mutant, cell lines with high copy number exhibited lower levels of complementation. High concentrations of UL9 appear to
be inhibitory in an in vitro helicase assay utilizing DNA
substrates involving elements of the HSV-1 origin of replication (37). It is believed that the inhibitory effect of the overexpressed UL9 is
mediated mainly by the origin-specific DNA binding function of UL9,
since transfection with the C-terminal domain of UL9 and wild type
infectious DNA can severely reduce the efficiency of plaque formation
(34, 40, 66). Moreover, when a mutant, UL9-OB, carrying an insertion
mutation (RIRA) in the C-terminal origin-binding domain (Fig. 1) known
to disrupt origin binding activity was cotransfected with wild type
infectious DNA, no inhibition was observed (40, 66). The inhibitory
properties of the overexpressed wild type UL9 are consistent with a
model in which HSV-1 DNA replication occurs in two stages (28).
According to this model, early in infection viral replication is
origin-dependent and occurs by a
When plasmids bearing UL9 motif I, motif II, and motif VI
mutant genes were transfected together with wild type infectious DNA,
the reduction of the number of viral plaques was even greater (Table
II) than was observed with the wild type, i.e. they are transdominant (dominant negative). In this report we showed that the
same mutants were able to dimerize, but their helicase activity was
completely abolished. The transdominance can thus be explained at least
in part by these biochemical characteristics. The mutants are able to
dimerize; therefore, the majority of UL9 present in the cell will
consist of dimers of mutant proteins. Small amounts of UL9 wild
type/mutant heterodimers (presumably nonfunctional) and even smaller
amount of wild type UL9 homodimers will also be present. Since the
mutant homodimers bind the origin of replication as well as wild type
and the mutant UL9 is expressed in huge excess, the majority of the
origins of replication will be bound by helicase negative mutant
homodimers. Therefore, the
The motif III mutant protein is a dimer with wild type levels of
helicase activity. In plaque reduction assays it appears as a
functional equivalent of wild type UL9, and it behaves as wild type in
transdominance assay (neutral). Another neutral mutant is motif IV,
which has been shown to aggregate (Fig. 3). At high protein
concentrations this mutant does not bind the origin DNA as efficiently
as wild type. Weaker origin binding may reduce inhibition compared with
the transdominant mutants resulting in a neutral phenotype in the
transdominance assay.
The motif V mutant is potentiating as judged by the increased number of
plaques in plaque reduction assay (Table II). In a previous study, the
motif V mutant protein could not be detected in transfected mammalian
cells (39). In this study experiments in insect cells revealed the
presence of N-terminal fragments only (data not shown), suggesting that
the C-terminal domain is degraded. The instability of the G354A mutant
protein may be due to conformational changes in the UL9 molecule
resulting in exposure of a protease target site and subsequent
cleavage. Currently, experiments are underway to understand the
potentiating phenotype of the motif V mutant.
In conclusion, we have characterized the biochemical properties of
several UL9 helicase motif mutants. Our results correlate with the
previously observed genetic phenotypes in in vivo
complementation and plaque reduction assays. The mutations of conserved
residues in the helicase motifs of UL9 result in impairment of the
ATPase of UL9 to different extents but do not significantly affect the ability of the protein to dimerize and bind specifically the origin of
replication. Mutations in all helicase motifs but motif III (T214S)
abolish the helicase function of UL9. Our results indicate that the
helicase function of UL9 is vital for the HSV-1 replication in
vivo. This finding together with the fact that UL9 is involved in
multiple protein-protein interactions with other replication proteins
(UL29/ICP8/SSB (67), UL42, a subunit of the HSV-1 DNA polymerase (68),
UL8, a member of the heterotrimeric replicative helicase-primase
complex (69), and pol VI of UL9 have been
isolated, and most of them fail to complement a UL9 null
virus in vivo (Martinez R., Shao L., and Weller S. (1992)
J. Virol. 66, 6735-6746). In addition, mutants in
motifs I, II, and VI were found to be transdominant (Malik, A. K.,
and Weller, S. K. (1996) J. Virol. 70, 7859-7866). Here we present the characterization of the biochemical
properties of the UL9 helicase motif mutants. We report
that mutations in motifs I-IV and VI affect the ATPase activity, and
all but the motif III mutation completely abolish the helicase
activity. In addition, mutations in these motifs do not interfere with
UL9 dimerization or the ability of UL9 to bind the HSV-1 origin of replication. Based on the similarity of the helicase motif sequences between UL9 and UvrB, another superfamily II member with
helicase-like activity, we were able to map the UL9
mutations on the structure of the UvrB protein and provide an
explanation for the observed phenotypes. Our results indicate that the
helicase function of UL9 is indispensable for viral replication,
supporting the hypothesis that UL9 is essential for unwinding the HSV-1
origin of replication in vivo. Furthermore, the data
presented provide insights into the mechanism of transdominance of the
UL9 helicase motif mutants.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-phosphates, whereas a conserved glutamate from
motif II is thought to activate a water molecule involved directly in
catalysis of the ATP hydrolysis (14, 17).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells were used for plasmid amplification.
70 °C. All solutions used
for protein purification contained 1 mM DTT, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride and 10 mM sodium bisulfite.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Domain structure of UL9. The UL9 protein
is thought to be composed of two domains. The helicase domain (residues
1-534) is presented as an open box and the origin-binding
domain (residues 535-851) is represented by a gray box. The
black boxes represent the helicase motifs (numbered
MI-MVI). The sites of mutations in each helicase motif used
in this study are shown, as is the OB mutation, a four-amino acid
insertion (RIRA) after residue 591 (66).
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Fig. 2.
Purified UL9 wild type and mutant
proteins. Wild type (WT) and mutant UL9 proteins were
purified from insect cells infected with recombinant baculoviruses as
described under "Experimental Procedures." Two micrograms of each
protein were resolved on a 10% SDS-gel and stained with Coomassie
Blue. Life Technologies, Inc., prestained markers were used as
molecular weight standards, and their positions are depicted on the
left.
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Fig. 3.
All helicase motif mutants, except motif IV,
are able to dimerize. Wild type and mutant UL9 proteins were
purified from insect cells infected with recombinant baculoviruses as
described under "Experimental Procedures." Fractions from the
Superose HR gel filtration column (Amersham Pharmacia Biotech) were
subjected to Western blot analysis with anti-UL9 rabbit polyclonal
serum R250. UL9 wild type (WT) and mutant proteins
(MI-MVI) peak in fractions 23-25, corresponding to a
dimeric state. Mutant protein MIV is aggregated and peaks in fractions
15-16. Gel filtration molecular mass markers (Sigma) are as
follows: apoferritin (443 kDa), amylase (200 kDa), alcohol
dehydrogenase (150 kDa), BSA (66 kDa), and carbonic anhydrase (29 kDa)
are depicted above the Western blot strips.
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Fig. 4.
Comparison of the overall protein
conformation of wild type and mutant UL9 proteins by limited
proteolysis. Wild type (WT) and mutant UL9 proteins
were purified from insect cells infected with recombinant baculoviruses
as described under "Experimental Procedures." Equal amounts of each
UL9 protein species were subjected to limited proteolysis with
proteinase K for time intervals of 0, 15, 30, and 60 min. The fate of
the N- and C-terminal proteolytic fragments was followed by Western
blot analysis. A, Western blot analysis with anti-UL9
antibody R250, recognizing the C terminus of UL9. The two major
C-terminal proteolytic fragments are marked with asterisks.
B, Western blot with anti-UL9 antibody 17B, recognizing the
N terminus of UL9. Lanes 13, 14, and 27 in
A and lanes 1 and 14 in B
contain the wild type protein sample at 15-min time point as a
reference. Note that the sensitivity of the 17B antibody is lower than
that of R250. Life Technologies, Inc., prestained markers were used as
molecular weight standards, and their positions are depicted on the
right.
1 in the absence of ssDNA and with a rate
constant of 27.1 min
1 in the presence of
ssDNA. M13 ssDNA stimulated wild type ATPase activity ~9-fold (Table
I). These findings are consistent with previously published reports (25). The intrinsic ATPase activity of the
motif III mutant (kcat 2.64 min
1) was comparable to the wild type
(kcat 2.96 min
1),
whereas it is almost completely abolished for the motif II mutant
(kcat 0.27 min
1). The
motif VI mutant retained 70% of the wild type intrinsic ATPase
activity, motif I retained 50%, and motif IV retained 30% (Fig.
5C compare the black bars). A more significant
effect was seen on the ssDNA-dependent ATPase activity. All
mutants retained only 25% or less of the wild type ssDNA-stimulated
ATPase activity (Fig. 5C, compare the gray bars).
All mutants except motif II exhibited lower than wild type stimulation
by ssDNA (Table I and below). The Km values for ATP
and M13 ssDNA were determined in the context of ssDNA-stimulated ATPase
activity. The values calculated for wild type (Table I) were comparable to values reported previously (50). Surprisingly, the
Km(ATP) value for motif I was found to
be 8-fold lower than wild type suggesting higher affinity for ATP
binding (see "Discussion"). Our efforts to assess directly the
effect of the helicase motif mutations on the ATP binding ability of
UL9 by UV-cross-linking failed due to extensive UL9 degradation in the
course of UV irradiation (data not shown). The Km
ssDNA for motif II is 5-fold lower than that for wild type (Table I).
The Vmax of the ssDNA-stimulated ATPase reaction
was reduced as little as 3-fold for the motif III mutant and as much as
10-fold for the motif I mutant (Table I). Although aggregated, the
motif IV mutant retained some ATPase activity (Fig. 5 and Table I),
confirming that the aggregation is most likely due to local
conformational changes perhaps caused by difficulties in accommodating
a bulkier tryptophan residue instead of phenylalanine. In summary,
mutations in all helicase motifs except motif III significantly
affected the ATPase function of UL9. The motif III mutant protein
exhibited wild type levels of the intrinsic ATPase activity and only a
moderate reduction of the ssDNA stimulated ATPase activity.
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Fig. 5.
Mutations in the UL9 helicase motifs affect
the intrinsic and ssDNA-stimulated ATPase activity. Wild type and
mutant UL9 proteins were purified from insect cells infected with
recombinant baculoviruses and assayed for ATPase activity as described
under "Experimental Procedures." A, time course of
intrinsic ATPase activity of wild type UL9 (WT, black
squares) and mutant proteins: MI (black diamonds), MII
(black triangles), MIII (open diamonds), MIV
(open squares), and MVI (open circles).
B, time course of ssDNA (M13)-stimulated ATPase activity of
wild type and mutant UL9. The symbols used are as described in
A. C, plot of the rate constants,
kcat (min 1), for the
intrinsic (black bars) and the ssDNA-stimulated ATPase
activity (gray bars). The error bars reflect the
S.D. calculated from three independent experiments.
Kinetic parameters of ssDNA-stimulated ATP hydrolysis
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Fig. 6.
Mutations in all helicase motifs except motif
III abolish the helicase activity of UL9. Wild type
(WT) and mutant UL9 proteins were purified from insect cells
infected with recombinant baculoviruses, and helicase assays were
performed as described under "Experimental Procedures."
A, each lane was loaded with one-half of the reaction mix
(30 µl) containing 1 nM helicase substrate and 200 nM enzyme. B, comparison of the helicase
activity of wild type and motif III mutant UL9. Each lane was loaded
with the entire helicase reaction (60 µl). Each reaction contained 5 mM ATP/Mg2+, 1 nM helicase
substrate, and 50 or 100 nM enzyme.
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Fig. 7.
UL9 helicase motif mutants bind specifically
OriS. Wild type (WT) and mutant UL9 proteins were
purified from insect cells infected with recombinant baculoviruses, and
a double membrane filter binding assay was performed as described under
"Experimental Procedures." Lanes 1-7 were loaded with
DNA eluted from the DEAE-cellulose membrane (unbound DNA). Lanes
8-14 were loaded with DNA eluted from the nitrocellulose membrane
(bound DNA). Lane 15 contains the input DNA. The lanes
designated with a dash contain no UL9 protein. Lanes
1-7 and 8-13 were loaded with equal counts/min and
therefore do not reflect the ability of each protein species to bind
OriS (see Fig. 8). 1-Kilobase pair and 50-base pair (bp) DNA
ladders (Life Technologies, Inc.) were used as a reference for DNA
fragment size.
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Fig. 8.
The abilities of wild type and mutant UL9
proteins to bind OriS are comparable. Wild type (WT) and mutant
UL9 proteins were purified from insect cells infected with recombinant
baculoviruses, and a double membrane filter binding assay was performed
as described under "Experimental Procedures." All binding reactions
contain 0.4 nM labeled OriS fragment. % bound DNA was
plotted with 100% representing % bound DNA for wild type UL9. The
gray bars represent binding reactions with 1 nM
enzyme; the black bars represent a binding reaction with 4 nM enzyme, and the white bars represent a
binding reaction with 10 nM enzyme. % bound DNA is defined
as 100 × (cpm nitrocellulose membrane)/(cpm nitrocellulose
membrane + cpm DEAE-cellulose membrane).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 9.
Mapping of the UL9
helicase motif mutants on the structure of UvrB, a superfamily II
helicase with homologous motif sequences. A, sequence
alignment of the UL9 and UvrB (B. caldotenax)
helicase motifs. Identical residues are highlighted in red;
hydrophobic residues (V, L, I, F, M, W, and Y)
are highlighted in green; small amino acids (G
and A) are highlighted in blue; negatively
charged (E and D) are highlighted in
yellow; and chemically similar pairs (S/T and
N/Q) are highlighted in gray. B, a ribbon diagram
of the UvrB (B. caldotenax) structure in a complex with ATP
(coordinates courtesy of Dr. C. Kisker, State University of New York,
Stony Brook). The helicase motifs are colored as follows: motif I,
yellow; motif II, orange; motif III,
cyan; motif IV, green; motif V,
magenta; and motif VI, royal blue. The ATP
molecule is colored in red. The arrows indicate
the UvrB residues that correspond to the mutations introduced in
UL9 (displayed in sticks). The UvrB residue
number is followed by the corresponding UL9 residue number and the
substitution mutation studied. The UvrB structure was displayed using
Molmol computer software.
-phosphate of ATP (11). In UL9, the substitution of Lys-87 with
alanine removes a positively charged long side chain. The alanine side
chain would not be able to coordinate the phosphate of the ATP, which
would explain the observed defects in UL9 ATPase and helicase
activities. One might imagine that the defects are due to impaired ATP
binding since the coordination of the phosphates is abolished.
Surprisingly, the Km value for ATP as seen in the
ATPase assay (Table I) was 8-fold lower than that for wild type
suggesting higher affinity for ATP binding. The same phenomenon was
observed for the NS3 (21) and the PcrA helicases (14). This apparent
discrepancy is not well understood. Soultanas et al. (14)
suggested that the lower Km(ATP) may
reflect "changes in rate constants associated with conformational changes or product release."
mechanism (stage I).
At the later times after infection, replication proceeds in an
origin-independent manner via a recombination-driven or rolling circle
mechanism (stage II). It is possible that if UL9 remains bound to the
origin of replication late in infection, it inhibits progression to
stage II. In agreement with this model, studies with UL9
temperature-sensitive mutants showed that UL9 is essential
for the early stages of HSV-1 replication and appears to be dispensable
for the later ones (31).
Summary of the properties of wild type and mutant UL9
-type replication (stage I) is likely to
be inhibited. In addition, the progression from stage I to II
replication would be inhibited by the mutant UL9 molecules remaining
bound at the origin of replication by a mechanism similar to the
inhibition by overexpression of wild type UL9 described above.
(70)) confirm the role of UL9 as a key player
in HSV-1 replication. Our data support a model in which UL9 is anchored
to the origin of replication via its origin-binding domain and, in
concert with ICP8 (36, 37, 71), unwinds the origin, creating a
replication bubble. The recruitment of heteromeric helicase/primase and
HSV-1 polymerase completes the assembly of the viral replication fork
(72-74), allowing replication to proceed.
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ACKNOWLEDGEMENTS |
---|
We thank all members of the Weller laboratory and Dr. S. Eisenberg for helpful discussions of the manuscript. We thank Dr. M. Challberg and Dr. D. Tenney for providing reagents used in this study. We also thank Dr. A. Malik for the generation pP10-based recombinant baculoviruses and Dr. C. Kisker for providing the coordinates of UvrB-ATP complex.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant AI21747.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.
To whom correspondence should be addressed: Dept. of Microbiology,
University of Connecticut Health Center, 263 Farmington Ave.,
Farmington, CT 06030. Tel.: 860-679-2310; Fax: 860-679-1239; E-mail:
Weller@NSO2.uchc.edu.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M007743200
2 In this paper the term "helicase motifs" is used to refer to motifs shared between large number of superfamily II members. It should be noted, however, that many SFII members have not been shown experimentally to exhibit helicase activity despite the presence of these "motifs."
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ABBREVIATIONS |
---|
The abbreviations used are: SFI, superfamily I; SFII, superfamily II; HSV-1, herpes simplex virus, type 1; ssDNA, single-stranded DNA; PMSF, phenylmethylsulfonyl fluoride; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; BSA, bovine serum albumin.
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1. | Subramanya, H. S., Bird, L. E., Brannigan, J. A., and Wigley, D. B. (1996) Nature 384, 379-383[CrossRef][Medline] [Order article via Infotrieve] |
2. | Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M., and Waksman, G. (1997) Cell 90, 635-647[Medline] [Order article via Infotrieve] |
3. | Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P., and Blinov, V. M. (1989) Nucleic Acids Res. 17, 4713-4730[Abstract] |
4. |
Korolev, S.,
Yao, N.,
Lohman, T. M.,
Weber, P. C.,
and Waksman, G.
(1998)
Protein Sci.
7,
605-610 |
5. | Bird, L. E., Subramanya, H. S., and Wigley, D. B. (1998) Curr. Opin. Struct. Biol. 8, 14-18[CrossRef][Medline] [Order article via Infotrieve] |
6. | Marians, K. J. (1997) Structure 5, 1129-1134[Medline] [Order article via Infotrieve] |
7. | Soultanas, P., and Wigley, D. B. (2000) Curr. Opin. Struct. Biol. 10, 124-128[CrossRef][Medline] [Order article via Infotrieve] |
8. | Kim, J. L., Morgenstern, K. A., Griffith, J. P., Dwyer, M. D., Thomson, J. A., Murcko, M. A., Lin, C., and Caron, P. R. (1998) Structure 6, 89-100[Medline] [Order article via Infotrieve] |
9. |
Cho, H. S.,
Ha, N. C.,
Kang, L. W.,
Chung, K. M.,
Back, S. H.,
Jang, S. K.,
and Oh, B. H.
(1998)
J. Biol. Chem.
273,
15045-15052 |
10. | Yao, N., Hesson, T., Cable, M., Hong, Z., Kwong, A. D., Le, H. V., and Weber, P. C. (1997) Nat. Struct. Biol. 4, 463-467[Medline] [Order article via Infotrieve] |
11. |
Theis, K.,
Chen, P. J.,
Skorvaga, M.,
Van Houten, B.,
and Kisker, C.
(1999)
EMBO J.
18,
6899-6907 |
12. |
Machius, M.,
Henry, L.,
Palnitkar, M.,
and Deisenhofer, J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11717-11722 |
13. | Benz, J., Trachsel, H., and Baumann, U. (1999) Structure Fold Des. 7, 671-679[Medline] [Order article via Infotrieve] |
14. | Soultanas, P., Dillingham, M. S., Velankar, S. S., and Wigley, D. B. (1999) J. Mol. Biol. 290, 137-148[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Lin, C.,
and Kim, J. L.
(1999)
J. Virol.
73,
8798-8807 |
16. | Schulz, G. E. (1992) Curr. Opin. Struct. Biol. 2, 61-67[CrossRef] |
17. | Story, R. M., and Steitz, T. A. (1992) Nature 355, 374-376[CrossRef][Medline] [Order article via Infotrieve] |
18. | Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S., and Wigley, D. B. (1999) Cell 97, 75-84[Medline] [Order article via Infotrieve] |
19. |
Graves-Woodward, K. L.,
Gottlieb, J.,
Challberg, M. D.,
and Weller, S. K.
(1997)
J. Biol. Chem.
272,
4623-4630 |
20. |
Brosh, R. M., Jr.,
and Matson, S. W.
(1996)
J. Biol. Chem.
271,
25360-25368 |
21. | Kim, D. W., Kim, J., Gwack, Y., Han, J. H., and Choe, J. (1997) J. Virol. 71, 9400-9409[Abstract] |
22. |
Dillingham, M. S.,
Soultanas, P.,
and Wigley, D. B.
(1999)
Nucleic Acids Res.
27,
3310-3317 |
23. | Carmichael, E. P., Kosovsky, M. J., and Weller, S. K. (1988) J. Virol. 62, 91-99[Medline] [Order article via Infotrieve] |
24. |
Bruckner, R. C.,
Crute, J. J.,
Dodson, M. S.,
and Lehman, I. R.
(1991)
J. Biol. Chem.
266,
2669-2674 |
25. | Fierer, D. S., and Challberg, M. D. (1992) J. Virol. 66, 3986-3995[Abstract] |
26. | Abbotts, A. P., and Stow, N. D. (1995) J. Gen. Virol. 76, 3125-3130[Abstract] |
27. | Weir, H. M., Calder, J. M., and Stow, N. D. (1989) Nucleic Acids Res. 17, 1409-1425[Abstract] |
28. | Weller, S. K. (1995) in The DNA Provirus: Howard Temin's Scientific Legacy (Cooper, G. M. , Temin, R. G. , and Sugden, B., eds) , pp. 189-213, American Society for Microbiology, Washington, D. C. |
29. | Challberg, M. (1997) in DNA Replication in Eucaryotic Cells (DePamphilis, M. L., ed) , pp. 721-751, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
30. |
Lehman, I. R.,
and Boehmer, P. E.
(1999)
J. Biol. Chem.
274,
28059-28062 |
31. | Blumel, J. B. M. (1995) J. Gen. Virol. 76, 3119-3124[Abstract] |
32. | Stow, N. D. (1992) J. Gen. Virol. 73, 313-321[Abstract] |
33. | Stow, N. D., Hammarsten, O., Arbuckle, M. I., and Elias, P. (1993) Virology 196, 413-418[CrossRef][Medline] [Order article via Infotrieve] |
34. | Perry, H. C., Hazuda, D. J., and McClements, W. L. (1993) Virology 193, 73-79[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Skaliter, R.,
and Lehman, I. R.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10665-10669 |
36. |
Lee, S. S.,
and Lehman, I. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2838-2842 |
37. |
He, X.,
and Lehman, I. R.
(2000)
J. Virol.
74,
5726-5728 |
38. | Makhov, A. M., Boehmer, P. E., Lehman, I. R., and Griffith, J. D. (1996) EMBO J. 15, 1742-1750[Abstract] |
39. | Martinez, R., Shao, L., and Weller, S. K. (1992) J. Virol. 66, 6735-6746[Abstract] |
40. | Malik, A. K., and Weller, S. K. (1996) J. Virol. 70, 7859-7866[Abstract] |
41. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Stuhl, K. (eds) (1990) Current Protocols in Molecular Biology , pp. 10.8.1-10.8.16, John Wiley & Sons, Inc., New York |
42. | Malik, A. K., Shao, L., Shanley, J. D., and Weller, S. K. (1996) Virology 224, 380-389[CrossRef][Medline] [Order article via Infotrieve] |
43. | Beynon, R. J. (1989) in Proteolytic Enzymes: A Practical Approach (Benyon, R. J. , and Bond, J. S., eds) , pp. 189-239, IRL Press at Oxford University Press, Oxford |
44. | Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. (1979) Anal. Biochem. 100, 95-97[Medline] [Order article via Infotrieve] |
45. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 4.21-4.32 and 10.51-10.53, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
46. | Crute, J. J., Mocarski, E. S., and Lehman, I. R. (1988) Nucleic Acids Res. 16, 6585-6596[Medline] [Order article via Infotrieve] |
47. | Wong, I., and Lohman, T. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5428-5432[Abstract] |
48. | Guarino, L. A., and Smith, M. W. (1990) Virology 179, 1-8[Medline] [Order article via Infotrieve] |
49. |
Elias, P.,
Gustafsson, C. M.,
Hammarsten, O.,
and Stow, N. D.
(1992)
J. Biol. Chem.
267,
17424-17429 |
50. | Earnshaw, D. L., and Jarvest, R. L. (1994) Biochem. Biophys. Res. Commun. 199, 1333-1340[CrossRef][Medline] [Order article via Infotrieve] |
51. | Theis, K., Skorvaga, M., Machius, M., Nakagawa, N., Van Houten, B., and Kisker, C. (2000) Mutat. Res. 460, 277-300[Medline] [Order article via Infotrieve] |
52. |
Lindahl, T.,
and Wood, R.
(1999)
Science
286,
1897-1905 |
53. | Hall, M. C., and Matson, S. W. (1999) Mol. Microbiol. 34, 867-877[CrossRef][Medline] [Order article via Infotrieve] |
54. | Brosh, R. M., Jr., and Matson, S. W. (1995) J. Bacteriol. 177, 5612-5621[Abstract] |
55. | Hall, M. C., Ozsoy, A. Z., and Matson, S. W. (1998) J. Mol. Biol. 277, 257-271[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Hall, M. C.,
and Matson, S. W.
(1997)
J. Biol. Chem.
272,
18614-18620 |
57. | Moolenaar, G. F., Visse, R., Ortiz-Buysse, M., Goosen, N., and van de Putte, P. (1994) J. Mol. Biol. 240, 294-307[CrossRef][Medline] [Order article via Infotrieve] |
58. | Wardell, A. D., Errington, W., Ciaramella, G., Merson, J., and McGarvey, M. J. (1999) J. Gen. Virol. 80, 701-709[Abstract] |
59. | Moore, K. J., and Lohman, T. M. (1994) Biochemistry 33, 14565-14578[Medline] [Order article via Infotrieve] |
60. | Moore, K. J., and Lohman, T. M. (1994) Biochemistry 33, 14550-14564[Medline] [Order article via Infotrieve] |
61. |
Porter, D. J.
(1998)
J. Biol. Chem.
273,
14247-14253 |
62. |
Porter, D. J.,
Short, S. A.,
Hanlon, M. H.,
Preugschat, F.,
Wilson, J. E.,
Willard, D. H., Jr.,
and Consler, T. G.
(1998)
J. Biol. Chem.
273,
18906-18914 |
63. |
Chao, K.,
and Lohman, T. M.
(1990)
J. Biol. Chem.
265,
1067-1076 |
64. |
Murata, L. B.,
and Dodson, M. S.
(1999)
J. Biol. Chem.
274,
37079-37086 |
65. | Malik, A. K., Martinez, R., Muncy, L., Carmichael, E. P., and Weller, S. K. (1992) Virology 190, 702-715[CrossRef][Medline] [Order article via Infotrieve] |
66. | Arbuckle, M. I., and Stow, N. D. (1993) J. Gen. Virol. 74, 1349-1355[Abstract] |
67. |
Boehmer, P. E.,
and Lehman, I. R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8444-8448 |
68. | Monahan, S. J., Grinstead, L. A., Olivieri, W., and Parris, D. S. (1998) Virology 241, 122-130[CrossRef][Medline] [Order article via Infotrieve] |
69. | McLean, G. W., Abbotts, A. P., Parry, M. E., Marsden, H. S., and Stow, N. D. (1994) J. Gen. Virol. 75, 2699-2706[Abstract] |
70. | Lee, S. S., Dong, Q., Wang, T. S., and Lehman, I. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7882-7886[Abstract] |
71. |
Boehmer, P. E.,
Dodson, M. S.,
and Lehman, I. R.
(1993)
J. Biol. Chem.
268,
1220-1225 |
72. | Liptak, L. M., Uprichard, S. L., and Knipe, D. M. (1996) J. Virol. 70, 1759-1767[Abstract] |
73. | Lukonis, C. J., and Weller, S. K. (1997) J. Virol. 71, 2390-2399[Abstract] |
74. |
Burkham, J.,
Coen, D. M.,
and Weller, S. K.
(1998)
J. Virol.
72,
10100-10107 |