From the Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030
Received for publication, November 6, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the
products of the UL5, UL52, and UL8
genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains
several conserved motifs found in primases; however, the
contributions of each subunit to the biochemical activities of the
subcomplex are not clear. In this work, the DNA binding properties of
wild type and mutant subcomplexes were examined using single-stranded,
duplex, and forked substrates. A gel mobility shift assay indicated
that the UL5-UL52 subcomplex binds more efficiently to the
forked substrate than to either single strand or duplex DNA. Although
nucleotides are not absolutely required for DNA binding, ADP stimulated
the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and
adenosine 5'-O-(thiotriphosphate) stimulated the binding to
a forked substrate. We have previously shown that both subunits contact
single-stranded DNA in a photocross-linking assay (Biswas, N., and
Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked
substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked
substrate when 5-iododeoxyuridine was located within the duplex
portion. Photocross-linking experiments with subcomplexes containing
mutant versions of UL5 and wild type UL52 indicated that the integrity
of the ATP binding region is important for DNA binding of both
subunits. These results support our previous proposal that UL5 and UL52
exhibit a complex interdependence for DNA binding (Biswas, N., and
Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active
role in helicase activity than had previously been thought.
DNA helicases catalyze the transient unwinding of
dsDNA1 to form ssDNA using
the energy of NTP hydrolysis. Helicases are essential in many
biological processes including replication, recombination, transcription, and DNA repair and have been isolated from prokaryotes, eukaryotes, and viruses. The helicase-primase complex of herpes simplex
virus type 1 (HSV-1) is a heterotrimeric complex composed of the
products of the UL5, UL52, and UL8 genes (1). All three genes are
essential for viral DNA replication (2-7). The UL5-UL52-UL8 complex
possesses primase, ssDNA-dependent NTPase, and 5' to 3' DNA
helicase activities (1, 8-11). The HSV-1 helicase-primase complex can
be isolated from insect cells that have been simultaneously infected
with recombinant baculoviruses that express each of the three subunits
(9). A subassembly consisting of the UL5 and UL52
gene products also exhibits all the enzymatic activities of the
holoenzyme in vitro (12). The UL5 protein contains seven conserved motifs found in all members of Superfamily 1 (SF1) helicase proteins (13). The UL52 protein contains several conserved motifs found
in other primases (14, 15). Neither UL5 nor UL52 appears to possess any
enzymatic activities when expressed alone (9, 12). The UL8 gene product
does not exhibit any enzymatic activities (10, 12) but can stimulate
both the helicase and primase activities of the helicase-primase
complex (16-19). Furthermore, UL8 may facilitate the entry of the
heterotrimer into the nucleus of infected cells (20, 21).
Although the molecular details of the mechanism of DNA unwinding is
unknown for any helicase, it is likely that the unwinding reaction
requires the coupling of several events such as ATP binding, ATP
hydrolysis, single strand and double strand DNA binding, and translocation along the DNA. Many helicases function as multimers such
as dimers (e.g. Escherichia
coli Rep (22, 23)) or hexamers (e.g.
helicases of T4 and T7 bacteriophages (24, 25) and SV40 large T antigen
(26, 27)). Although it has been suggested that oligomeric structures
provide multiple DNA binding sites, which are required for helicase
action (28), it appears that at least two helicases, E. coli
DNA helicase II and Bacillus stearothermophilus PcrA, are
active as monomers (29, 30). Three models to explain the mechanism of
helicase activity have been proposed. The inchworm model posits that
conformational changes caused by binding and hydrolysis of ATP cause a
helicase monomer to "inch" along the DNA (30, 31). Monomeric
helicases would presumably contain at least two nonidentical DNA
binding sites on each monomer. The rolling model, which is based on the
dimeric Rep protein, posits that a helicase must act as (at least) a
dimer and that each subunit of the dimer can bind to either ssDNA or
duplex DNA (23). According to this model, a helicase rolls along the
DNA with alternating subunits binding first to ds then to ssDNA. A
third model proposed for the hexameric helicases posits that the core
of the hexameric unit provides a channel through which a single strand
of DNA can be threaded (32-34). The protein would move along one
strand with alternating subunits responsible for ATP hydrolysis. To
distinguish between these models and to understand the mechanism of
helicase action, it will be necessary to obtain more detailed
information about how helicases contact DNA. Two members of SF1
helicases, Rep and PcrA, have been crystallized in the presence of DNA
(30, 35). The crystal structure of the E. coli Rep helicase
bound to ssDNA, and ADP revealed putative contact residues for ssDNA on
the protein (35); however, many of these assignments have not been
confirmed by genetic analysis.
Previous DNA binding studies revealed that the UL5-UL52 subcomplex
binds to ssDNA more effectively than to dsDNA and that the minimum
length of ssDNA that can bind and stimulate its ATPase activity is
about 12 nucleotides (36). Herein we show that the UL5-UL52 subcomplex
binds much more efficiently to a forked substrate than to either ss or
dsDNA. The fact that the HSV-1 helicase is part of a multiprotein
complex complicates the analysis of the DNA binding sites of the
individual subunits. We have previously shown that both subunits can
contact single-stranded DNA in a photocross-linking assay (37).
Moreover, we have shown a complex interdependence on both subunits for
DNA binding, in that a mutation in the putative Zinc binding domain of
the UL52 subunit has drastic effects on the ability of UL5 to
cross-link single-stranded DNA. In this paper we have taken two
approaches to study the interaction of the UL5-UL52 subcomplex with
DNA. Cross-linking studies using forked substrates with substitutions
of deoxyuridine (dIU) in three different positions indicate that the
UL5 and UL52 subunits contact the forked substrates at different
positions; UL5 appears to contact DNA near the fork, whereas UL52
appears to contact the ss tail of the forked substrate. Neither subunit
appears to directly contact dsDNA. In a second approach, we performed
DNA binding and cross-linking assays on a series of UL5 mutants whose mutations lie in conserved helicase motifs shared by other SF1 members
(38, 39). The results confirm a complex interdependence between the two
subunits and indicate that the UL52 subunit may play a more active role
in helicase activity than had previously been thought. Furthermore,
these studies suggest that the HSV-1 helicase-primase may act as a
monomer (one heterotrimer per replication fork) and favor the inchworm
model for the mechanism for helicase activity.
Reagents--
Supplemented Graces's medium and 10%
Pluronic®F were purchased from Life Technologies, Inc.
Fetal calf serum was obtained from Atlanta Biologicals.
Penicillin-streptomycin solution, ampicillin, phenylmethylsulfonyl
fluoride, leupeptin, and pepstatin were purchased from Sigma. The 20-ml
HiLoad 16/10 SP-Sepharose fast flow column was from Amersham
Pharmacia Biotech. Inc. The 12-ml Uno Q (Q-12) column was from Bio-Rad.
The 25-ml Superose 12 HR column was from Bio-Rad. Radiolabeled
nucleotides were purchased from Amersham Pharmacia Biotech.
Substituted oligonucleotides were synthesized from Cruachem. A
polyclonal antibody (1248) directed against the C-terminal 10 amino
acids of UL52 was a kind gift from Dr. Mark Challberg (National
Institutes of Health, Bethesda, MD).
Buffers--
Buffer A consists of 20 mM HEPES (pH
7.6), 1.0 mM dithiothreitol (DTT), 10 mM sodium
bisulfite, 5 mM MgCl2, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 1 µg/ml
pepstatin, and 2 µg/ml aprotinin. Buffer B contains 20 mM
HEPES, pH 7.6, 1.0 mM DTT, 10% (v/v) glycerol, and 0.5 mM EDTA. All buffers were passed through a 0.22-µm filter
and degassed before use.
Cells and Viruses--
Spodoptera frugiperda
(Sf9) cells were maintained at 27 °C in Graces's insect
medium containing 10% fetal calf serum, 0.33% lactalbumin
hydrolysate, 0.33% yeastolate, 0.1 mg/ml streptomycin, and 100 units/ml penicillin. The recombinant Autographa californica nuclear polyhedrosis baculovirus expressing HSV-1 UL5 was
generously provided by Dr. Robert Lehman (Stanford University School
of Medicine, Stanford, CA). The recombinant baculovirus expressing UL52
was a kind gift from Dr. Nigel D. Stow (Medical Research Council
Virology Unit, Glasgow, United Kingdom). The recombinant baculovirus
expressing UL8 was generously provided by Dr. Mark Challberg
(National Institutes of Health). Baculovirus recombinants harboring UL5
motif mutant genes, AcUL5G102V (motif I), BacUL5-DE249,250AA (motif
II), BacUL5-G290S (motif III), AcUL5R345K (motif IV), AcUL5-G815A
(motif V), and BacUL5-Y836A (motif VI) were described previously (39).
Viral stocks were amplified in Sf9 cells grown in suspension as
described previously (39). Stocks were titered by determining the
volume of viral stock, which gave the maximum level of recombinant
protein expression on 1 × 106 Sf9 cells at
48 h post-infection.
Protein Expression and Purification--
2 liters of Sf9
cells were grown in suspension at 27 °C in Graces's insect medium
as described previously (39). The wild type and variant UL5-UL52
subcomplexes were purified essentially as described earlier with an
additional gel filtration step. Cells were dounced using 15 strokes of
a tight fitting pestle in Buffer A, and the cytosolic extracts were
clarified by centrifugation at 35,000 × g for 30 min.
UL5-UL52 subcomplexes were precipitated from the cytosolic extract by
the addition of an equal volume of Buffer B containing 0.2 M NaCl and 2 M ammonium sulfate and incubation
on ice for 4 h. The resultant protein pellets were resuspended in
Buffer B containing 0.1 M NaCl and dialyzed against the
same buffer. The dialyzed sample was loaded onto an SP-Sepharose column
equilibrated with Buffer B containing 0.1 M NaCl, and the column was washed with 5 column volumes of the equilibration buffer. Fractions containing the UL5-UL52 subcomplex were identified by SDS
polyacrylamide gel electrophoresis. The UL5-UL52 subcomplex elutes from
the column in the void volume. Pooled fractions from the SP-Sepharose
column were loaded onto a 12-ml Uno Q column equilibrated with Buffer B
containing 0.1 M NaCl. The column was washed with five
column volumes of Buffer B containing 0.1 M NaCl, and the
protein was eluted using a 185-ml linear gradient of Buffer B
containing 0.1-1 M NaCl. Pooled fractions from the Uno Q
column were concentrated using a centrifugal concentrator with a 10-kDa cut-off (MicrosepTM, Pall Filtron) and loaded onto a
25-ml Superose 12 HR column equilibrated with Buffer B containing 0.1 M NaCl. The fractions containing the peak activities were
pooled, concentrated, and frozen at DNA Substrates--
An 18-mer of oligo(dT), PCdT18(5),
with a dIU substitution at the 5th T from the 5' end was
synthesized by Cruachem and end-labeled with
[ Gel Mobility Shift Assay--
Gel mobility shift assays were
essentially performed as described previously (39). The reaction
mixture (25 µl) contained 20 mM Na+ HEPES (pH
7.6), 1 mM DTT, 0.1 mg/ml bovine serum albumin, 10% glycerol, 5 mM MgCl2, 1.2 pmol (molecules) of
the DNA substrates labeled with [ Photocross-linking--
Photocross-linking experiments
were performed essentially as described previously with 1.2 pmol of the
indicated DNA substrate molecules and 4 pmol of UL5-UL52 subcomplex in
20 mM Na+ HEPES (pH 7.6), 1 mM DTT,
0.1 mg/ml bovine serum albumin, 10% glycerol, and 5 mM
MgCl2 (37). The samples were incubated on ice for 10 min
before irradiation. An IK series He-Cd laser (IK 3302R-E,
KIMMON; Kimmon Electric Co., Ltd.) was used to achieve monochromatic
325-nm light. The laser beam output was 34 milliwatts measured with a
power meter, Mentor MA10, Scientech® (Scientech, Inc., Boulder, CO).
Samples were irradiated in a methacrylate cuvette (catalog number
14-385-938; Fisherbrand) at room temperature. At different time points
aliquots were withdrawn, boiled for 5 min in SDS-PAGE loading buffer,
and subjected to SDS-PAGE on an 8% gel. The gels were dried and
exposed to film at UL5-UL52 Subcomplex Binds to a Forked Substrate More Efficiently
than to either Single-stranded or Double-stranded DNA--
Previous
studies with the UL5-UL52 subcomplex indicated a preference for ss
versus dsDNA; in a filter-binding experiment the subcomplex
bound ssDNA about 5-fold more effectively than it did dsDNA (36). We
previously showed that the UL5-UL52 subcomplex could bind a forked
substrate generated by the annealing of partially complementary
oligonucleotides (39). Here we compare the binding efficiencies of
UL5-UL52 to the forked substrate and to single- and double-stranded DNA
using a mobility shift experiment. Fig. 1
shows that the UL5-UL52 subcomplex can bind forked, ss, and dsDNA (Fig.
1, lanes b, i, and p, respectively).
The gel shift data indicate that the binding of the UL5-UL52 subcomplex
to a forked substrate is at least 8-fold higher than to ssDNA and is at
least 35-fold higher than to dsDNA. Addition of UL8 to the binding
reaction resulted in a supershift to a slower migrating species (Fig.
1, lanes c, j, and q); in the case of
the forked substrate (Fig. 1, lane c), the supershifted band
is somewhat smeared, perhaps reflecting the complex interactions
exhibited by UL5-UL52 with forked DNA (see below). Quantification of
the gel shift data indicates that the UL8 stimulates the binding
of UL5-UL52 to both forked (1.8-fold) and ss (2.2-fold) DNA substrates (Table I). To determine whether
nucleotide di- and triphosphates play a role in the DNA binding
properties of UL5-UL52, the binding of the subcomplex with ss and
forked DNA substrates was tested in the presence of ATP, ADP, and
ATP Cross-linking of UL5-UL52 to a Forked Substrate--
We have
previously used a photocross-linking assay to show that both UL5 and
UL52 subunits of the wild type UL5-UL52 subcomplex can contact a short
ss oligomer (37). During replication, the helicase-primase would
presumably contact a replication fork consisting of double- and
single-stranded DNA; we therefore initiated cross-linking studies using
a series of forked substrates shown in Fig.
2. A He-Cd light source that emits at 325 nm was used to photocross-link the UL5-UL52 subcomplex to a
32P end-labeled forked substrate in which dIU was
substituted for one of the thymidine residues. In FS B, dIU was placed
in the ss portion of the substrate at a position 7 nucleotides from the 5' end of the lower strand (Fig. 2). The UL5-UL52 subcomplex was cross-linked to a 5' 32P-labeled single strand
oligonucleotide (Fig. 3A,
lane a) or to a 5' 32P-labeled forked substrate
(FS B) (Fig. 3A, lane b). As previously reported
(37), when the subcomplex was cross-linked to the single strand
oligonucleotide, two labeled bands were observed by SDS-PAGE, one
migrating at ~100 kDa corresponding to UL5 and a slower band migrating at 120 kDa corresponding to UL52 (Fig. 3A,
lane a). When the UL5-UL52 subcomplex was cross-linked to FS
B, however, slower migrating bands were observed (Fig. 3A,
lane b); the uppermost band migrates at a position
corresponding to ~220 kDa and a lower set of smeared bands, which may
contain two or more species migrating at a position corresponding to
170-195 kDa. A time course of binding in which the UL5-UL52 subcomplex
was incubated with FS B irradiated for varying lengths of times was
performed to determine whether the pattern of cross-linked bands
changes with time. Fig. 3B shows that the time of
irradiation correlates with the amount of cross-linked material and
that the pattern of bands, a 220-kDa band and two or more bands
migrating between 170 and 195 kDa, remains constant throughout the
experiment.
To further characterize the cross-linking to a forked substrate,
substrates were generated that contain dIU at various positions (Fig.
2). FS A does not contain any substitutions, FS B was described above
and contains a substitution entirely within the single-stranded portion
of the substrate, FS C contains a dIU substitution within the duplex
portion of the forked substrate at the 27th position from the 5' end of
the lower labeled strand (11 base pairs from the ss/ds junction), and
FS D contains dIU substitution at the 15th position from the 5' end of
the lower strand very close to the ss/ds junction (Fig. 2). In a gel
shift assay, it is apparent that the UL5-UL52 subcomplex can bind both
FS B and FS C with equal efficiencies (Fig.
4A, lanes b and
d). However, the cross-linking experiment shown in Fig.
4B demonstrates that UL5-UL52 can be cross-linked to FS B
much more efficiently than it can be cross-linked to FS C (Fig.
4B, compare lanes a and b to
lanes c and d). Quantification of the
cross-linked bands indicates that cross-linking was 4.4-fold more
efficient to FS B than FS C at the 15-min time point and 6.2-fold more
efficient at the 30-min time point. This result suggests that although
FS C can be bound to the subcomplex as assessed by the gel mobility
assay, neither UL5 nor UL52 are located in close proximity to the
duplex portion of the substrate. In the experiment shown in Fig.
5, forked substrates B and D were compared. By gel mobility assay, both substrates were bound with equal
efficiency (Fig. 5A, lanes b and d).
In this case the UL5-UL52 subcomplex could be cross-linked to FS B and
FS D with more or less equal efficiency (Fig. 5B); however,
the mobility of the bands cross-linked to FS D was very different from
those cross-linked to FS B. In this case two or more bands that migrate
in the range of 115-180 kDa were observed (see below). The observation
that the subcomplex cross-links to both FS B and FS D but is unable to
cross-link to FS C suggests that the subcomplex is bound to the
single-stranded rather than the duplex region of the forked substrate.
DNase1/S1 Nuclease Digestion of Cross-linked
Species--
SDS-PAGE analysis of the FS B and FS D cross-linked
species revealed the presence of multiple radioactive bands that
migrate more slowly than UL5 and UL52 (Figs. 4 and 5). The forked
substrate itself has a molecular mass of 30 kDa, and the slow
mobilities of the cross-linked species may be a result of binding
multiple substrate molecules to either UL5 or UL52. To characterize the composition of the slower migrating cross-linked species, they were
treated with DNase1 and S1 nuclease for increasing amounts of time.
Treatment with nucleases is expected to degrade the DNA substrates
allowing identification of the proteins present in the high molecular
weight complexes. Fig. 6A
demonstrates that after treatment of the FS B cross-linked
material with nucleases, two radiolabeled bands appear that migrate at
the same position as UL52 and UL5. At 5, 10, and 20 min of digestion,
the strongest band corresponds to UL52. By 2 h, the signals
corresponding to both UL5 and UL52 disappeared almost entirely (Fig.
6A, lane g). In Fig. 6B, the FS D
cross-linked material was treated with nucleases for various periods of
time. At the 20- and 40-min time points, a signal corresponding to UL5
was predominant (Fig. 6B, lanes d and
e). This experiment suggests that the high molecular weight cross-linked bands represent bound forms of UL5 and UL52 to a forked
substrate. Furthermore, the signals obtained after partial nuclease
digestion suggest that UL5 preferentially cross-links to FS D, which
contains the dIU substitution at a position very close to the ss/ds
junction, whereas UL52 preferentially cross-links to FS B, which
contains the dIU residues close to the 5' end of the ssDNA tail.
Immunoblot Analysis of Cross-linked Complexes--
Immunoblotting
was used to confirm the identity of the slower migrating species
observed when the UL5-UL52 subcomplexes are cross-linked to forked
substrates B and D (Figs. 4B and 5B, described above). UL5-UL52 subcomplexes cross-linked either to a single strand
substrate or forked substrate or not cross-linked were subjected to
SDS-PAGE in duplicate; one-half of the gel was processed for
autoradiography, and the other half was subjected to immunoblot analysis with antisera raised against either UL5 or UL52. Fig. 7A shows the autoradiogram of
the cross-linked samples; as in Fig. 3, slower migrating bands of
~170-195 and 220 kDa are seen when the UL5-UL52 subcomplex is
cross-linked to FS B (Fig. 7A, lane 2). In these
cross-linking experiments, only a small proportion of the UL5 and UL52
proteins are actually cross-linked; therefore, it is expected that
immunoblotting will detect uncross-linked UL5 and UL52. As predicted,
in the experiments shown in Fig. 7B, antisera against UL5
(lanes 4, 5, and 6) reacts primarily
with a band corresponding to uncross-linked UL5, although a weak band corresponding to UL52 is also observed, presumably because of cross-reactivity of the antisera with UL52. Antisera against UL52 (Fig.
7C, lanes 9, 10, and 11)
primarily reacts with a band corresponding to UL52 and a weak band
corresponding to UL5, again probably because of cross-reactivity.
Interestingly, in the material cross-linked to the forked substrate,
three slower migrating bands corresponding to 170-195 kDa (marked with
an *) were detected with the UL52 antibody (Fig. 7C,
lane 10). No slower migrating bands were detected in the ss
cross-linked sample or in the uncross-linked protein sample (Fig.
7C, lanes 9 and 11, respectively).
This result indicates that UL52 is present in the complexes
cross-linked to FS B. We cannot rule out that some UL5 is also present;
however, the predominant signal appears to be UL52. In the experiment
shown in Fig. 8, the UL5-UL52 subcomplex
was cross-linked to FS D. Fig. 8A shows the autoradiogram of
the subcomplex cross-linked to ssDNA or FS D or uncross-linked (Fig.
8A, lanes 3, 2, and 1,
respectively). With the UL5 antibody, a strong UL5 uncross-linked band
was seen in all three lanes (Fig. 8B, lanes
5-7). A faint band migrating at UL52 was seen in the sample
cross-linked to ssDNA; this is probably because of cross-reactivity of
UL52 with the UL5 antibody (Fig. 8B, lane 7).
Several more intense slower migrating bands could be detected in the
material cross-linked to FS D, indicating that UL5 is present in the
cross-linked material (Fig. 8B, lane 6). With the
UL52 antibody, only the uncross-linked UL52 was detected (Fig.
8C, lanes 9-11). Thus it appears that, as
described above, UL52 is cross-linked preferentially to FS B, and UL5
is cross-linked preferentially to FS D; however, we cannot rule out
that these complexes also contain small amounts of the other
subunit.
To confirm that the high molecular weight cross-linked species
represent complexes containing UL5 and UL52, as the complexes with
single strand DNA clearly do, competition experiments with single-stranded 48-mer DNA oligonucleotide (either unlabeled or labeled) were also performed. This experiment indicates that the slower
migrating bands of the complex with FS B disappear in the presence of
unlabeled ssDNA (data not shown). If labeled ssDNA is used as the
competitor, the high molecular weight bands are decreased in intensity,
and bands corresponding to UL5 and UL52 bound to ssDNA are observed,
although the UL52 band is stronger (data not shown). These results
confirm that the high molecular weight species seen with the FS B
substrate contained both the UL5 and the UL52 subunits, although UL52
was predominant as demonstrated above.
Cross-linking of Mutant UL5-Wild Type UL52 Subcomplexes to ss and
Forked Substrates--
The DNA binding sites within the
helicase-primase complex have not been mapped. We previously reported
the isolation and characterization of UL5 mutants bearing mutations in
the conserved motifs shared among SF1 members (39). In that study, we
found that mutant subcomplexes were able to bind forked substrates
using a gel shift assay as well or better than wild type subcomplexes;
however, this assay reflects the DNA binding of the whole complex, and it was not possible to determine the individual contributions of either
subunit. For instance, if one of the UL5 mutant proteins was defective
for DNA binding, it may not have been apparent using this assay,
because a defect may have been masked by the binding of the UL52
subunit. To characterize UL5 binding more directly and map regions of
UL5 responsible for contacting DNA, we tested subcomplexes containing
mutant UL5 proteins and wild type UL52 for their ability to cross-link
various substrates. In the experiment shown in Fig.
9, wild type and motif mutant
subcomplexes were irradiated for 10 and 30 min by a He-Cd laser in the
presence of the labeled single-stranded 18-mer oligo(dT) substrate,
which contains one dIU substitution. SDS-PAGE and subsequent
autoradiography revealed that the UL5 subunit with a mutation in motif
I (Fig. 9A, lanes c and d) or motif
III (Fig. 9A, lanes g and h) is
somewhat defective in cross-linking to ssDNA compared with wild type
(Fig. 9A, lanes a and b). Fig.
9B is a Coomassie-stained gel showing that approximately the
same amount of protein was loaded into each cross-linking reaction.
Quantification demonstrates that the DNA binding ability of the motif I
mutant UL5 subunit was 1.7-fold less than wild type, and the motif III
mutant was 1.6-fold less than wild type (Fig. 9C). The other
UL5 mutant proteins cross-link to ssDNA with approximately wild type
efficiency. Fig. 9A also shows that in some of the mutant
subcomplexes, the UL52 subunit fails to bind ssDNA effectively. For
instance, in the subcomplexes containing the motif I mutation (Fig.
9A, lanes c and d), the motif II
mutation (Fig. 9A, lanes e and f), or
the motif III mutation (Fig. 9A, lanes g and
h), cross-linking to ssDNA of the UL52 subunit was
significantly lower than wild type. Quantification indicates that UL52
subunit binding is decreased 6.5-fold in motif I, 3.5-fold in motif II,
and 2.7-fold in motif III (Fig. 9C). Motif IV, motif V, and
motif VI exhibited wild type levels of cross-linking of both the UL5
and UL52 subunits to ssDNA substrate (Fig. 9, A and C).
In the experiment shown in Fig. 10, we
asked whether the ability of motif mutant subcomplexes to cross-link
forked DNA was different from their ability to cross-link the ssDNA
substrate described above. Six different UL5 helicase motif mutants
(motifs I, II, III, IV, V, and VI) were compared with wild type for
their ability to cross-link to forked substrate B (Fig. 10,
A and B). The overall cross-linking efficiency as
measured by adding the intensities of the two radiolabeled bands was
slightly (1.3-fold) lower in the motif I mutant subcomplex compared
with wild type (Fig. 10A, compare lanes b and
d). The motif V and motif VI mutant subcomplexes showed
slightly higher efficiencies than wild type (Fig. 10A,
lanes j and l, respectively), whereas motif III
and motif IV mutant proteins showed 2.4- to 2.8-fold higher
cross-linking efficiencies than wild type (Fig. 10, lanes f
and h, respectively). The motif II mutant binds with
approximately wild type efficiency to the forked substrate (Fig.
10B, compare lanes c and d to
a and b). Thus the ability of the mutant
subcomplexes to cross-link to a single-stranded DNA substrate differs
considerably from their ability to bind to forked substrates; even
subcomplexes with defects in ssDNA binding appear to be stabilized on
forked substrates.
In this paper we have studied DNA binding of the HSV-1
helicase-primase by analyzing the substrate preferences of the
helicase-primase complex and by determining the binding properties of
subcomplexes containing various mutant forms of the UL5 subunit.
Several observations were made: 1) UL5-UL52 binds preferentially to a
forked substrate over ss or dsDNA substrate in a mobility shift assay.
2) Although nucleotides are not absolutely required for DNA binding,
ADP stimulates the binding of UL5-UL52 to ssDNA, whereas ATP, ADP, and
ATP Substrate Preferences and Effects of Nucleotides on DNA
Binding--
Previous reports indicated that HSV-1 UL5-UL52 can bind
ssDNA 5-fold more effectively than ds plasmid DNA by filter binding assay (36). To our knowledge, this paper presents the first comparison
between forked, ss, and dsDNA substrates for the HSV helicase-primase.
The observation that DNA binding to the forked substrate is much better
than to the single strand substrate can be explained in at least three
non-mutually exclusive ways: 1) It is possible that the
helicase-primase needs to bind first to single-stranded DNA to be able
to recognize dsDNA. In other words, the helicase needs to be loaded
onto dsDNA. Thus, the previously observed low affinity for dsDNA may
reflect the fact that the enzyme can only bind dsDNA after it has
contacted ssDNA. 2) Each subunit (UL5 and UL52) appears to have the
capacity to contact DNA individually as determined by cross-linking
studies to single strand substrates (37); however, binding to the
forked substrate may reflect cooperativity between binding site(s) on
each of the subunits resulting in more stable binding to the forked
substrate. 3) The structure of the forked substrate itself may act to
promote binding of the subcomplex. For instance, the presence of a
joint region between the ds and ss regions of the substrate may provide a binding surface that greatly stabilizes the binding of one or both subunits.
Models for the mechanism of helicase unwinding have been proposed that
make predictions concerning the types of DNA contacts a helicase is
expected to make with its substrate and its stoichiometry of binding.
According to the rolling model, each helicase subunit must be able to
bind ssDNA, as well as dsDNA, but not both at the same time (28).
According to the inchworm model, a monomer may need to bind ss and
dsDNA at the same time at least during a portion of the reaction cycle
(30). In the hexameric helicases, the helicases are proposed to contact
ssDNA primarily. Thus, the binding affinities and stoichiometry of
binding have important implications for the mechanism of helicase
action. The stoichiometry of binding of the UL5-UL8-UL52 complex at the
replication fork is not known; however, it is possible that the
UL5-UL8-UL52 complex exists either as a monomer (one heterotrimer) or
as a dimer (two molecules of the heterotrimer). A dimer of trimers
might be expected to function either as a dimer consistent with the
rolling mechanism or as hexamer. Two lines of evidence appear to rule
out the rolling mechanism for the HSV-1 helicase-primase. First, the
UL5-UL52 subcomplex does not bind efficiently to dsDNA as would be
predicted by the rolling mechanism. Second, genetic analysis from our
laboratory suggest that UL5 does not function as a dimer or higher
order structure like a hexamer; mutants in motifs I and II that abolish helicase and ATPase activity do not exert a transdominant effect on
wild type UL5 function.2 This
is in contrast to mutants in motifs I and II of UL9 that inactivate helicase activity and are strongly transdominant; for UL9,
the potent transdominant activity appears to be due at least in part to
the ability of UL9 to dimerize (40, 41). Another reason to suspect that
the HSV-helicase-primase is unlikely to function as a hexamer is that
only superfamily 3 and family 4 helicases have been shown to function
as hexameric helicases (reviewed in Refs. 42 and 43). In summary, the
genetic and DNA binding evidence lead us to favor the inchworm
mechanism for this helicase-primase complex. According to the inchworm
model, each monomeric unit would possess at least two binding sites.
This implies either that UL5 has two or more binding sites or that one
of the other subunits, most likely UL52, contributes to helicase
activity by providing a second DNA binding site. We favor the later
possibility as it is consistent with existing data (discussed below).
In this study we also confirmed and extended previous reports on
effects of nucleotides on the DNA binding activities of the UL5-UL52
subcomplex. Healy et al. (36) had previously reported that
the ssDNA binding activity of UL5-UL52 could be stimulated 1.7-fold in
presence of ADP. In this paper we confirm this result, and in addition
we show that ATP, ADP, and ATP UL5-UL52 Interactions with Forked
Substrates--
Photocross-linking experiments were designed to
elucidate the contributions made by the individual subunits to DNA
binding and to determine which part of the ss and ds junction of a
replication fork is occupied by each subunit of the UL5-UL52 complex.
The slow mobility of subcomplexes cross-linked to either FS B or FS D
(Figs. 4B and 5B) indicates that there are two to
three substrate molecules bound to each enzyme complex. These data
suggest that there may be more than one DNA binding site per
subcomplex. A combination of experiments including DNase treatment,
Western blot analysis, and competition experiments indicate that the
higher molecular weight radiolabeled bands are indeed composed of the UL5 and UL52 subunits. Furthermore, these experiments suggest that UL5
preferentially cross-links at a position close to the ss/ds junction,
whereas UL52 preferentially cross-links within the ss region of the
forked substrate.
Within the HSV-1 helicase-primase, UL5 has long been assumed to be the
helicase and UL52 to be the primase; however, several lines of evidence
suggest a complex interdependence on both subunits for the activities
of the subcomplex. For instance, Barrera et al. (46)
analyzed an intertypic helicase-primase complex consisting of a UL5
subunit from HSV-1 and a UL52 subunit from HSV-2. This subcomplex
exhibited decreased helicase and primase activities and diminished
neurovirulence, indicating that small structural changes in the UL5
subunit could also affect primase activity. Furthermore, we previously
showed that a mutation in the putative Zn binding region of the UL52
subunit abolished not only primase activity but also ATPase and
helicase activities (37). In addition, both UL5 and UL52 subunits
within the mutant subcomplex were totally defective in cross-linking to
ssDNA (37). To explain these and other observations discussed below, we
propose that UL52 may play a more important role than previously
recognized in the helicase activity of the subcomplex. It is possible
that binding of UL52 to ssDNA may be necessary to load the UL5 subunit.
Alternatively, UL52 may play an even more active role in the helicase
mechanism by providing a second DNA binding site necessary during the
unwinding reaction.
Mutations in the Helicase Motifs of UL5--
We previously
reported the biochemical analysis of mutants in conserved residues in
the motifs of UL5. We found that motif I is directly involved in ATP
binding and/or hydrolysis and that motif II appears to be required for
coupling of DNA binding to ATP hydrolysis. Residues in motifs III, IV,
V, and VI are involved in the coupling of ATP hydrolysis and DNA
binding to the process of DNA unwinding (39). The defects in ATPase
activity in the UL5 mutants can be explained in light of the recently
solved crystal structure of two other SF1 family helicases, Rep and
PcrA, which exhibit a remarkable degree of similarity to each other
(30, 35, 47). Both contain two recA-like domains arranged such that the
conserved helicase motifs all lie along a cleft between them. This
arrangement has led to the suggestion that helicase activity may be
carried out through conformational changes within the molecule in
response to ATP binding, ATP hydrolysis, and binding of DNA (30, 35,
47). The severe defects in ATPase activity exhibited by the UL5
mutations in motifs I and II are consistent with a role in ATP binding
and hydroylsis. Furthermore, the lack of coupling between ATPase and
helicase activities of mutations in motifs III, IV, V, and VI (39) can
be explained by the position of these motifs along the cleft between
the two recA like domains. Our results support the proposal that the
conserved motifs play a role in mediating conformational changes within
the molecule in response to DNA and nucleotide binding.
The crystal structure of Rep and PcrA in the presence of
single-stranded DNA has also been reported (30, 35). In both cases, the
ssDNA was found to bind along the top of the recA-like domains, and
residues from motifs Ia, III, and V were shown to contact ssDNA. To
confirm the predictions made from the structural information about Rep
and PcrA for UL5, the motif mutants described above were analyzed for
their ability to bind various substrates. Cross-linking data with ss
substrates indicated that subcomplexes containing motif I mutations are
defective not only in UL5 but also UL52 binding. This result was
somewhat surprising, because motif I is not physically located near the
putative ssDNA binding cleft in the other SF1 family helicases. The
binding defects of this mutant may be explained by the fact that ATP is
an allosteric effector of the DNA binding activity of the enzyme. The
structural integrity of the ATP binding domain of UL5 may be essential
for DNA binding of the entire complex. Alternatively, the integrity of
ATP binding domain could be required for the proper folding or
stability of UL5. However, the fact that all mutant UL5 proteins retain
the ability to interact with UL52 and UL8 suggest that the mutation in
motif I does not dramatically alter the overall structure of the protein.
In subcomplexes containing the UL5 motif II mutant, UL5 was able to
cross-link ssDNA and forked DNA with wild type efficiency; however, the
UL52 subunit was defective in ssDNA cross-linking. This may also be
because of the fact that ATP is an allosteric effector of the
entire complex; perhaps the subcomplex is affected by the inability of
UL5 to bind and/or hydrolyze ATP. Interestingly, despite the apparent
defect in the ability of UL52 to bind ssDNA, the motif II UL5 mutant
subcomplex exhibits wild type levels of primase activity but no
helicase activity (39). Thus, the DNA binding activity of UL52 in
mutant subcomplexes does not necessarily correlate with primase
activity supporting the notion that at least some of the DNA binding
ability of UL52 contributes to helicase activity, not primase.
The defects in the ability of motif III mutant UL5 proteins to bind
ssDNA support the structural prediction that motif III interacts
directly with ssDNA in two other SFI family helicases (30, 35). This
result is consistent with a previous report showing that a motif III
mutant of DNA helicase II is unable to form a stable binary complex
with either DNA or ATP (48). Despite the fact that motif III
subcomplexes were defective for binding ssDNA, motif III and IV
subcomplexes could cross-link better than wild type to the forked
substrate. This result confirms our previous observation that these two
mutants were able to bind forked substrates 5- to 6-fold better than
wild type, respectively, in a gel shift assay (39). This result may
also reflect the fact that binding of the subcomplex to the forked
substrate can be stabilized even in the presence of mutant subunits
with decreased affinity for ssDNA. In general, subcomplexes containing
mutants defective in binding ssDNA (Fig. 9) were less defective in
their ability to bind to forked substrates either by cross-linking
(Fig. 10) or by gel shift assays (33), again supporting the notion that
subcomplexes can be stabilized on the forked substrate.
Interestingly, Motif III and IV subcomplexes also exhibited dramatic
increases, 36- and 9-fold, in primase activity (39). This rather
drastic effect on primase activity may reflect a complex regulation of
helicase and primase activities within the helicase-primase complex. A
single heterotrimeric helicase-primase complex bound to DNA would not
be expected to carry out helicase and primase activities
simultaneously; helicase is believed to move in the 5' to 3' direction
along the lagging strand template, whereas primase activity necessarily
occurs in the 3' to 5' direction along the template. It is possible
that that there may be competition between the helicase site and the
primase site for binding to DNA. Thus, mutation of the helicase motifs
of the UL5 polypeptide may disrupt binding of the UL5 helicase subunit
to DNA, increasing the likelihood of DNA binding at the primase active
site. Alternatively, as suggested above, when helicase is active, UL52
may act as a second DNA binding subunit contributing to helicase
activity, precluding it from binding to the primase recognition site.
When helicase activity is abolished, as in a helicase-defective mutant, UL52 is free to bind primase recognition sites thus resulting in
increased primase activity and ability to bind to forked substrates. In
the assays used in this paper, a 5' to 3' helicase (like the HSV-1
helicase-primase) would be expected to contact the lower strand of the
forked substrates shown in Fig. 2 during lagging strand synthesis,
whereas the primase would be expected to contact the upper strand.
Thus, the substrates were not optimized for looking at contacts between
primase and ssDNA within the forked substrates. To test our models
fully, however, it will be important to study forked substrates bearing
a dIU substitution on the top strand, as well as substrates that have
only a 5' or 3' tail instead of two ssDNA tails. The presence of the
preferred primase recognition site on one or the other tail will also
be tested.
In summary, in this paper we have taken two approaches to the study of
interactions between the helicase-primase subcomplex with DNA. We have
studied substrate preferences for the wild type version
helicase-primase subcomplex and have analyzed binding properties of UL5
motif mutants. The DNA binding data and the behavior of mutant
subcomplexes in cross-linking assays have lead to the suggestion that
the UL52 subunit may play a more active role in helicase activity than
had previously been thought. Our results are also consistent with the
inchworm mechanism for helicase activity for the HSV-1
helicase-primase. Further experiments will be required to test these models.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
-32P]ATP. Forked DNA substrate A was constructed
by heat denaturing and annealing 80 pmol of the helicase 48C/FS
oligonucleotide (5' CGAAAGTACGTTATTGCGACTGGCCGTCGCTCTACAACGTCGTGACTG 3')
radiolabeled at its 5' end with [
-32P]ATP and 80 pmol
of unlabeled 48FS oligonucleotide (5'
CAGTCACGACGTTGTAGAGCGACGGCCAGTCGGTTATTGCATGAAAGC 3'). The
underlined residues are complementary and create the duplex region of
the molecule. After annealing, the products were subjected to
electrophoresis on an 8% nondenaturing polyacrylamide gel, and the
forked substrate was purified by electroelution and ethanol
precipitation. Forked substrates (FS B, FS C, and FS D) were prepared
by annealing 80 pmol of each of the end-labeled 48C/FSM
oligonucleotide (5'
CGAAAGdIUACGTTATTGCGACTGGCCGTCGCTCTACAACGTCGTGACTG 3'), 48C/FSM27 oligonucleotide (5'
CGAAAGTACGTTATTGCGACTGGCCGdIUCGCTCTACAACGTCGTGACTG 3'), or 48C/FSM15 oligonucleotide (5'
CGAAAGTACGTTATdIUGCGACTGGCCGTCGCTCTACAACGTCGTGACTG 3'), respectively, to 80 pmol of the unlabeled 48FS oligonucleotide. In
FS B, the substitution is in the 7th position from the 5' end of the
lower (labeled) strand (see Fig. 2). In FS C, the dIU substitution is
within the duplex region (see Fig. 2), and in FS D, the substitution is
within the ss region of the lower (labeled) strand very near the
ss/dsDNA junction. The duplex DNA substrate was prepared in a similar
manner; 80 pmol of 32 S oligonucleotide
(5'CAGTCACGACGTTGTAGAGCGACGGCCAGTCG3') was annealed to the
complementary 32 CS oligonucleotide (5'CGACTGGCCGTCGCTC- TACAACGTCGTGACTG3').
-P32]ATP and 4 pmol
of the UL5-UL52 subcomplex with or without UL8 protein (12 pmol), ATP
(5 mM), ADP (5 mM), or ATP
S (5 mM). The reaction was allowed to proceed for 10 min on ice
and terminated by the addition of one-tenth of a volume of a loading
solution (80% glycerol, 0.1% bromphenol blue). Reaction products were
analyzed on a 4% nondenaturing acrylamide, 0.11% bisacrylamide gel at
150 V at 4 °C. The gels were dried and exposed to film at
70 °C.
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (Fig. 1). Single strand DNA binding was stimulated 1.6-fold in
the presence of ADP, and the binding of UL5-UL52 to forked substrate
was stimulated in the presence of ATP (1.4-fold), ADP (1.3-fold), and
ATP
S (1.5-fold) (Table I). In summary, it appears that the UL5-UL52
subcomplex binds much more efficiently to the forked substrate than to
either single-stranded or double-stranded DNA; the addition of UL8 or nucleotide cofactors exhibited modest but reproducible stimulatory effects on DNA binding of the subcomplex. The similar levels of stimulation observed with ADP, ATP, and ATP
S suggest that the binding of ATP but not its hydrolysis is important for optimal binding
of UL5-UL52 to the forked substrate.
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Fig. 1.
UL5-UL52 subcomplex binds preferentially to a
forked substrate compare with ss or dsDNA. The gel mobility shift
assay was performed using 1.2 pmol of the radiolabeled forked
(lanes a-g), ss (lanes h-n), or duplex
(lanes o-u) DNA substrates and 4 pmol of the UL5-UL52
subcomplex in the presence of 12 pmol of UL8 protein (lanes
c, j, and q), 5 mM ATP
(lanes d, k, and r), 5 mM
ADP (lanes e, l, and s), and 5 mM ATP S (lanes f, m, and
t). The samples were incubated for 10 min on ice and
analyzed by 4% nondenaturing polyacrylamide gel electrophoresis as
described under "Experimental Procedures." Lanes a,
h, and o represent the reaction in the absence of
protein. Lanes g, n, and u represent
the reaction containing only UL8 protein. no enz, no
enzyme.
Relative affinity of UL5-UL52 for forked and single strand oligo DNA
substrates in the presence and absence of UL8 and nucleotides
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Fig. 2.
Forked substrates. The substrates
(A, B, C, and D) were
constructed by annealing two partially complementary ss
oligonucleotides. The lower strand was radiolabeled at the
5' end with 32P in each case. FS B, FS C, and FS D contain
one dIU (represented by x) residue in a different position
of the molecule as described under "Experimental Procedures."
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Fig. 3.
Cross-linking of UL5-UL52 with ssDNA and
forked substrate. Cross-linking reactions were carried out using a
dIU-substituted ss oligo(dT), (A, lane a), or FSB
(A, lane b) in a methacrylate cuvette (light
path, 10 mm) at room temperature with an He-Cd laser emitting 34 milliwatts at 325 nm. Samples were removed after 30 min of irradiation.
Samples were boiled for 5 min in 1× SDS-PAGE loading buffer and
subjected to electrophoresis on a 8% SDS-PAGE gel, which was then
dried and exposed to film overnight at 70 °C. Arrows in
A indicate UL5 and UL52 cross-linked to ssDNA. The
approximate molecular mass of each radioactive band was calculated from
the standard graph of the 10-kDa protein ladder. In B a time
course experiment was carried out using FS B. Samples were removed
after 0 (lane a), 2 (lane b), 4 (lane
c), 10 (lane d), 30 (lane e), and 60 (lane f) min of irradiation (B).
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Fig. 4.
DNA binding of UL5-UL52 with FS B and FS
C. The gel shift assay (A) and the cross-linking assay
(B) were carried out essentially as described in legends for
Fig. 1 and Fig. 3 using forked substrate B (lanes a and
b) and forked substrate C (lanes c and
d). Lanes a and c (A)
represent the reaction in the absence of any protein. In the
cross-linking reaction, samples were taken out at the 15-min
(B, lanes a and c) and 30-min
(B, lanes b and d) time points.
Lane e represents the cross-linking reaction of UL5-UL52
with ss oligonucleotide.
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Fig. 5.
DNA binding of UL5-UL52 with FS B and FS
D. Gel shift assay (A) and cross-linking assay
(B) were carried out essentially as described in the legend
for Fig. 4 using forked substrate B (lanes a and
b) and forked substrate D (lanes c and
d). Lanes a and c (A)
represent the reaction in the absence of any protein. In cross-linking
reaction samples were taken out at the 15-min (B,
lanes a and c) and 30-min (B,
lanes b and d) time points.
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Fig. 6.
DNase1/S1 nuclease digestion of UL5-UL52-FS
cross-linked species produced radiolabeled UL5 and UL52 proteins.
Cross-linking reactions (0.15 ml) were carried out using 1.2 pmol of FS
B (A) or FS D (B) and 4 pmol of UL5-UL52 protein.
Samples were exposed for 30 min, and a 0.02-ml aliquot was withdrawn at
the 0-min (A and B, lane a) time
point. 10 µg of DNase1 and 18 units of S1 nuclease were added, and
the mixture was incubated at 37 °C. A 0.02-ml aliquot was withdrawn
at 5-min, 10-min, 20-min, 40-min, 60-min, and 2-h intervals
(A, lanes b-g, respectively). In B,
aliquots were taken at 5-, 10-, 20-, and 40-min intervals (lanes
b-e, respectively). Samples were boiled for 5 min in 1× SDS-PAGE
loading buffer and subjected to electrophoresis on a 8% SDS-PAGE gel,
which was then dried and exposed overnight at 70 °C to film.
UL5-UL52 cross-linked with ssDNA were shown in lane h and
i (A) and in lane f
(B).
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Fig. 7.
Detection of UL52 protein in UL5-UL52-FS B
cross-linked species by Western blot analysis. Cross-linking
reaction was carried out using FS B (A, lane 2;
B, lane 5; and C, lane 10)
and ssDNA (A, lane 1; B, lane
4; and C, lane 9) as described previously.
Samples were irradiated for 30 min, concentrated by a centricon, and
analyzed by SDS-PAGE. A, lane 3; B,
lane 6; and C, lane 11 represent a
control of uncross-linked protein. A represents the
autoradiogram of one-half of the gel. Samples from the other half of
the gel were transferred onto nitrocellulose and processed for anti-UL5
(B) or anti-UL52 (C) polyclonal antibody.
Arrows indicate the position of ss cross-linked or
uncross-linked UL52 and UL5. Lanes marked M
contain the 10-kDa protein ladder. The asterisk (*) indicates slower
migrating bands detected with anti UL52 antibody.
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Fig. 8.
Detection of UL5 protein in UL5-UL52-FS D
cross-linked species by Western blot analysis. The cross-linking
reaction was carried out as described in the legend for Fig. 7 using FS
D. A represents the autoradiogram of the gel. Samples from a
duplicate SDS-PAGE gel were transferred onto nitrocellulose and
processed for anti-UL5 (B) or anti-UL52 (C).
Arrows indicate the position of ss cross-linked UL52 and UL5
(which migrate identically to uncross-linked UL5 and UL52).
Lanes marked M contain the 10-kDa protein ladder.
The * indicates slower migrating bands detected with anti-UL5
antibody.
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Fig. 9.
SDS-PAGE analysis of the wild type and
helicase motif mutant (MI-MVI) subcomplexes
cross-linked with single-stranded DNA. Cross-linking reactions
were carried out in a methacrylate cuvette (light path, 10 mm) at room
temperature with an He-Cd laser emitting 34 milliwatts at 325 nm.
Samples were removed after 10 and 30 min of irradiation for wild type
(WT) and UL5 motif mutants (MI-MVI). Samples were
boiled for 5 min in 1× SDS-PAGE loading buffer and subjected to
electrophoresis on a 8% SDS-PAGE gel, which was then dried and exposed
overnight at 70 °C to film. Panel A represents the
autoradiogram, panel B represents the Coomassie-stained gel,
and panel C represents the quantification of cross-linking
data.
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Fig. 10.
SDS-PAGE analysis of wild type and mutant
subcomplexes cross-linked with FS B. Cross-linking reactions were
carried out as described before using 4 pmol of helicase motif mutant
subcomplexes and FS B. Motif I, motif III, motif IV, motif V, and motif
VI, along with wild type, are shown in A, and motif II,
along with motif III and WT, are shown in B. Samples were
cross-linked for 15 and 30 min and were analyzed by 8% SDS-PAGE. Motif
III is shown in both figures as an additional control to compare
binding ability of the motif II mutant subcomplex. Panel C
represents the quantification of cross-linking data from panel
A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S stimulates the binding to a forked substrate. 3) The UL5-UL52 subcomplex can be cross-linked to a forked substrate, and the composition of the resulting cross-linked species varies depending on
the position of the dIU substitution. When the substitution is within
the ss region of the substrate, UL52 is preferentially cross-linked;
however, when the substitution is near to the ss/ds junction, UL5
appears to be preferentially cross-linked. 4) UL5 proteins bearing
mutations in the conserved helicase motifs varied in the ability of
subcomplexes containing mutant UL5 and wild type UL52 to bind ss or
forked substrates. These results suggest that the ATP binding region is
important for DNA binding of both subunits and confirm our previous
finding of a complex interdependence between UL5 and UL52 subunits for
their DNA binding properties (discussed below).
S can stimulate the binding of the
protein to a forked substrate. Modulation of DNA binding affinity by
nucleotide cofactors have been reported for several helicases (23, 44,
45). Kinetic studies with the Rep helicase suggests that ADP favors
binding to ssDNA whereas a nonhydrolyzable ATP analog favors the
simultaneous binding to both ss and duplex DNA by the Rep dimer (23).
The behavior of HSV-1 helicase-primase appears to resemble the Rep
helicase in this respect, because ADP can stimulate the binding to both
forked substrates and ssDNA, but ATP and ATP
S stimulate the binding to the forked substrate only (Table I). Thus, binding of
ATP-Mg2+ but not its hydrolysis may be important for
optimal binding of helicase to the forked substrate. The binding of ATP
may allosterically regulate the affinity of the UL5-UL52 protein for
different types of DNA substrates. The binding of ATP and other
nucleotides may enhance the formation of protein-DNA complexes or
increase their stability once formed, or both.
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ACKNOWLEDGEMENTS |
---|
We thank members of our laboratory and Dr. Mark Challberg for helpful comments on the manuscript. We especially thank Boriana Marintcheva for assistance in figure preparation and helpful discussions.
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FOOTNOTES |
---|
* This investigation was supported by 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.
Present address: Dept. of Biology, University of California, 9500 Gilman Dr., San Diego, CA 92093-0366.
§ To whom correspondence should be addressed. Tel.: 860-679-2310; Fax: 860-679-1239; E-mail: weller@nso2.uchc.edu.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010107200
2 R. Zhou and S. K. Weller, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ds, double-stranded;
ss, single-stranded;
HSV-1, Herpes simplex virus type 1;
SF1, Superfamily 1;
dIU, 5-iododeoxyuridine;
DTT, dithiothreitol;
FS, forked substrate;
ATPS, adenosine
5'-O-(thiotriphosphate);
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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