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(Received for publication, January 5, 1995; and in revised form, May 25,
1995) From the
The herpes simplex virus type 1 (HSV-1) origin binding protein,
OBP, is a DNA helicase specifically stimulated by the viral single
strand DNA-binding protein, ICP-8. The stimulation is dependent on
direct protein-protein interactions between the C-terminal domain of
OBP,
It has been suggested on the basis of studies of the replication
of Escherichia coli and its phages that DNA replication is
carried out by multimeric machines(1, 2, 3) .
Numerous contacts between the proteins involved and the DNA substrate
can be expected to be established and broken during the dynamic
progression of the replisomes. The replication of herpes simplex virus
1 seems to be no exception. A multimeric replisome capable of rolling
circle replication has recently been isolated(4) . The events
leading to the formation of this replisome at the viral origins of
replication, oriS and oriL, are presumably initiated by the HSV-1 ( OBP is an
ATP-dependent DNA helicase encoded by the UL 9
gene(6, 7, 8) . It is a member of the
helicase superfamily 2(9) . A remarkable feature of this
protein is its ability to interact specifically with several proteins
in the putative HSV-1 replisome and also with different DNA
ligands(10, 11) . These interactions are in some
instances dependent on a co-factor, ATP, and they may also trigger ATP
hydrolysis. The interaction between OBP and double-stranded DNA has
the following characteristics. It is strongly sequence specific with an
approximate K OBP also binds to single-stranded DNA albeit
with lower affinity. This interaction results in a strong stimulation
of ATP hydrolysis(7, 18) . Biochemical studies have
identified a complex between the C-terminal DNA-binding domain of OBP
and the HSV-1 single strand DNA-binding protein ICP 8(10) .
Apparently, 30 amino acids at the C terminus of OBP are required to
form this complex(19) . This interaction causes an
approximately 10-fold stimulation of an ATP-dependent DNA helicase
activity(8, 18, 19) . The second component
of this complex, ICP 8, binds single-stranded DNA in a cooperative way.
The site occupied on single-stranded DNA has been estimated to be
between 12-40 nucleotides(20, 21, 22) .
The DNA-binding domain of ICP 8 is localized between the amino acid
residues 564 and
849(23, 24, 25, 26) . ICP 8 binds
one Zinc atom and contains one Zn-finger motif at positions
499-512(27) . ICP 8 is also a component of the HSV-1
replisome as isolated by ion-exchange chromatography and may therefore
bind to one or more of the UL30, UL42, UL5, UL8, or UL52 gene
products(4) . The biological role of ICP 8 is clearly to
stimulate DNA replication, but it has also been implicated in
homologous recombination(20, 28) . In fact, it has
recently been demonstrated that it can promote homologous pairing and
strand transfer(29) . We have now investigated how the
interaction between OBP and ICP 8 is controlled by DNA ligands. Our
results suggest a model where this interaction serves to position ICP 8
onto single-stranded DNA at an origin of replication.
The restriction fragments used in this work have been derived from
the plasmid series pORI and pORI(boxImut)(17) . pORI(wt)
contained an unaltered origin sequence. The numbering of the nucleotide
sequence is the same as before(17) . In the plasmid
pORI(boxImut), two point mutations were introduced in box I lowering
the affinity for OBP by two orders of
magnitude(15, 17) . The plasmids containing spacer
sequences of altered lengths pORI(+4),(-2),(-4),
and(-6) have been described(17) .
A BamHI fragment
containing the C-terminal domain of OBP was also cloned downstream from
the GST gene in the expression vector pGEX-2T (Pharmacia). Expression
of GST ICP 8 was purified from SF9 cells infected
with a recombinant baculovirus vector obtained from Dr. Nigel Stow, MRC
Virology Unit, Glasgow. The purification was performed as
described(10) . The purity of the proteins used throughout
this study was greater than 90% as estimated by SDS-polyacrylamide gel
electrophoresis. Protein concentrations were determined with the
bicinchonininic acid (BCA) protein assay reagent (Pierce) or from
Coomassie-stained SDS-gels calibrated by BSA standards.
Chromatography was performed on Superose 12 HR 10/30 (Pharmacia).
The column was equilibrated in 20 mM HEPES-NaOH, pH 7.6, 0.2 M NaCl, 1 mM DTT, and 10% glycerol. Chromatography
was performed at a flow rate of 0.2 ml/min at +4 °C. Fractions
of 0.2 ml were collected, and the protein content was analyzed by
SDS-polyacrylamide gelectrophoresis. The gels were stained with
Coomassie Brilliant Blue G-250 and dried. The content of ICP 8 and
His The recovery of the individual
components after chromatography was approximately 70%. The column
used was calibrated with the following proteins of known molecular
weights: myoglobin, 16,800; ovalbumin, 43,000; bovine serum albumin,
66,000; aldolase, 158,000; and ferritin, 440,000.
The binding of His
Figure 7:
The formation of complexes bewteen
His
The Stability of Complexes between His Chromatography of
free His
Figure 1:
Chromatography on Superose 12 of
complexes between His
When
His In
an experiment where His The binding of ICP 8 to a
single-stranded oligonucleotide was investigated in an experiment where
ICP 8 and T65 were mixed in equimolar proportions. The complex ICP
8/T65 eluted at an apparent molecular weight of 850,000 (Fig.1, a and b, and Table 1). The stoichiometry of
this complex will be further discussed below. The influence of DNA
ligands on the stability of the His
Figure 2:
The formation of a ternary complex between
ICP 8, His
Figure 3:
Digestion of ICP 8-T300 complexes with
micrococcus nuclease reveals a 14-nucleotide repeat. A uniformely
labeled synthetic polynucleotide T300 was mixed with saturating amounts
of ICP 8. The resulting complex was subjected to limited digestion by
micrococcus nuclease. The deproteinized DNA was analyzed on 6%
polyacrylamide sequencing gel. The autoradiographs of the dried gels
were analyzed by computing densitometry.
We have also investigated the
interaction between His
Figure 4:
His
Furthermore, in experiments employing quenching of
tryptophane fluorescens as a method of detecting an interaction between
His
Figure 5:
Heterodimers between His
Early in this series of investigations we noted
that the behavior of His We have previously noted that His
Figure 6:
His
Figure 11:
Models describing the His
Finally, we wanted to examine the effect of
single-stranded DNA on the formation complexes between ICP
8,His
Figure 8:
A single-stranded oligonucleotide prevents
ICP 8 from binding to a complex between His
Figure 9:
A DNase I footprint of a His
Figure 10:
ICP 8 is positioned at the AT-rich spacer
of oriS. Densitometric scanning profiles of the autoradiograph in Fig.6highlighting the protection of a phosphodiester bond
located in the AT-rich spacer sequence of oriS. Nucleotide 43 is marked
with an asterisk ((17) ). a, free oriS (Fig.6, lane 1). b, oriS and ICP 8 (Fig. 6, lane 2). c, oriS and His
We also investigated the
structure of the complex formed between ICP 8,His The synthesis of chromosomal DNA is carried out by
replication machines or replisomes(1, 2, 3) .
These multimeric machines are assembled in an orderly manner at unique
locations on DNA, and it has become an important objective to identify
the interactions that lead to the assembly of the replication apparatus
and keep it together. Less is known about how these interactions are
controlled in order to allow the dynamic progression of a replisome
along the DNA template. The replication of herpes simplex virus DNA
seems also to be carried by a machine. Recently, a complex consisting
of six proteins encoded by the virus has been isolated(4) .
This complex apparently contains the proteins encoded by the UL 5, 8,
29, 30, 42, and 52 genes, and it seems to be capable of carrying out
origin-independent DNA synthesis. It is assumed that the interactions
that lead to the formation of this replication machine are initiated by
the OBP at the viral origin of replication oriS. A considerable number
of these interactions have been identified. OBP can bind directly to
ICP 8, the product of the UL 29 gene, and the UL 8
protein(10, 11) . The latter is in addition a part of
the trimeric helicase-primase complex coded for by the UL 5, 8, and 52
genes(33) . The DNA polymerase forms specific contacts with the
UL 42
protein(34, 35, 36, 37, 38) .
In as much as ICP 8 is observed within the replication machine in the
absence of OBP, one can easily postulate additional functionally
important protein-protein interactions in which ICP 8 would take part.
We have here addressed the question how the interaction between OBP and
ICP 8 is affected by their DNA ligands. When the C-terminal DNA-binding
domain of OBP was used we observed a specific complex between these
proteins showing a stoichiometry of 1:1 in complete agreement with the
results of Boehmer and Lehman(10) . In the presence of a
specific duplex oligonucleotide containing the recognition sequence for
OBP, a complex containing all three components was observed. A
titration experiment suggested that the stoichiometry of this complex
was 1:1:1. However, when a single-stranded oligonucleotide was present
the interactions between His With this discussion in mind, one can
construct a more detailed model of the initial steps occurring during
the initiations of DNA replication at oriS (Fig.11b).
In this case we suggest that two dimers of OBP bind cooperatively to
oriS in an ATP-dependent way(15, 17) . A total of 4
ICP 8 molecules can be bound by this complex. Two of these are
positioned at the AT-rich spacer sequence. When this sequence becomes
unwound ICP 8 will leave OBP and attach to DNA. In order to position
the next ICP 8 molecules at the unwound region, we can imagine that OBP
carries out a rolling motion induced by ATP hydrolysis perhaps
analogous to the reaction performed by the rep
helicase(39, 40) . In this way two dynamic cycles are
coupled. In the first case the association and disassociation of ICP 8
and OBP is controlled by the availability of single-stranded DNA. The
second cycle is characterized by hydrolysis of ATP dependent on
single-stranded DNA. This model does not attempt to explain the
molecular motions that OBP has to perform, and it does not address the
role of the cooperative interactions between two OBP dimers. Are these
stable enough to keep the putative tetramer intact during the
continuous unwinding of DNA? Perhaps they only occur at oriS and will
be broken as soon as additional replication proteins, possibly UL 8,
enters the scene. It would also be of interest to see to what extent
the DNA ligand can influence protein-protein interactions in other
replication machines. It is, for example, interesting to note that the
interaction between the gene 32 and gene 61 proteins of bacteriophage
T4 might be influenced by the DNA ligand(41) . The eukaryotic
single strand DNA-binding protein RP-A interacts with the simian virus
40-encoded helicase large T-antigen(42) . One would like to
assume that this interaction could also be a part of a dynamic cycle.
Volume 270,
Number 32,
Issue of August 11, pp. 19028-19034, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
OBP, and ICP 8 (Boehmer, P. E., Craigie, M. C., Stow, N. D.,
and Lehman, I. R.(1994) J. Biol. Chem. 269,
29329-29334). We have now observed that this interaction is
dramatically influenced by the nature of the DNA ligand. Stable
complexes between OBP, ICP 8, and double-stranded DNA, presented
either as a specific duplex oligonucleotide or a restriction fragment
containing the HSV-1 origin of replication, oriS, can be detected by
gel chromatography and gel electrophoresis. In contrast, a
single-stranded oligonucleotide, oligo(dT), will
completely disrupt the complex between
OBP and ICP 8. We therefore
suggest that the interaction between OBP and ICP 8 serves to
position the single strand DNA-binding protein with high precision onto
single-stranded DNA at a replication fork or at an origin of DNA
replication.
)origin binding protein, OBP(5) . of 0.1 nM for the
specific recognition sequence
CGTTCGCACTT(12, 13, 14) . The binding of OBP
to two copies of this sequence in oriS is
cooperative(13, 14, 15, 16) . The
formation of OBP
oriS complexes is influenced by ATP and other
nucleotide co-factors as well as the phasing of the recognition
sequences(17) .
Plasmids, Restriction Fragments, and
Oligonucleotides
The duplex oligonucleotide PE 17/18 containing
the box I sequence of HSV-1 oriS was prepared as described
before(12) . A single-stranded 65-mer of oligodeoxythymidylate
denoted T65 was purchased from Scandinavian Gene Systems. A uniformly
labeled 300-mer of polydeoxythymidylate, T300, was synthesized with
terminal transferase using 1 pmol of a 15-mer of oligodeoxythymidylate
as primer and 360 pmol of [
-P]dTTP 80
Ci/mmol. T300 was purified by chromatography on Bio-Gel P10 (Bio-Rad).
Purification of His
A pET3A expression vector for the C-terminal DNA-binding
domain of OBP (OBP, GSTOBP, and ICP
8OBP) was modified by the insertion of a duplex
oligonucleotide introducing the amino acid sequence
MetSerHis
GlyThrGly at the N terminus. The cells transformed
with the pET3a/HisOBP plasmid were grown, induced, and harvested
as described previously(15) . A crude extract was prepared
following published procedures(13, 15) . Purification
in the absence of detergent was performed on a 50 ml of
Ni-NTA column (Qiagen) eluted with a 10-200
mM imidazole gradient in 0.1 M NaCl, 20 mM
Na
-HEPES, pH 7.4, 10% glycerol. Additional
purification could be achieved using chromatography on a column of
S-Sepharose (Pharmacia) developed by a linear gradient consisting of
0.15 M NaCl versus 1.0 M NaCl in 20 mM MOPS adjusted to pH 7.4 with NaOH.
OBP and subsequent purification was performed essentially as
described(30) .Chromatography of Protein
Complexes between HisDNA
ComplexesOBP, 2 nmol or 74 µg,
and ICP 8, 2 nmol or 256 µg, were formed by mixing equimolar
amounts of protein in 0.2 ml of the equilibration buffer described
below. Incubation was for 20 min on ice. The oligonucleotides PE 17/18,
2 nmol, or T65, 0.7 nmol, were then added as indicated and the
incubation was allowed to proceed another 10 min on ice. OBP in the fractions was determined using the computing
densitometer Molecular Dynamics 300A. The chromatographic profiles of
the radioactively labeled oligonucleotides PE 17/18 and T65 were
determined by scintillation counting.Digestion of Complexes between ICP 8 and T300 with
Micrococcus Nuclease
The reaction mixture, 20 µl, contained
0.9 fmol of T300 and 9 pmol of ICP 8 in 20 mM Tris-HCl, pH
7.5, 3 mM MgCl, 0.1 M NaCl, 0.1 mM EDTA, 1
mM DTT, and 10% glycerol. 0.2 ng of micrococcus nuclease
(Pharmacia) was added and incubated for 30 min at 37 °C. The
reaction was stopped by 5 µl of 25 mM EDTA and 0.02 mg/ml
sonicated calf thymus DNA. The samples were then extracted with phenol
and precipitated with ethanol. They were finally analyzed on a 6%
polyacrylamide sequencing gel.Gel Electrophoresis of Protein
The binding of HisDNA
ComplexesOBP and GSTOBP to the
duplex oligonucleotide PE 17/18 was analyzed on 7% polyacrylamide gels
containing acrylamide and bisacrylamide at a ratio of 80:1 using
Tris-glycine as the running buffer(17) . A reaction mixture of
10 µl contained 10 fmol of the radiolabeled duplex oligonucleotide
PE 17/18 1600 Ci/mmol and HisOBP and GSTOBP as indicated in
the figure legend. The incubation buffer was 0.38 M glycine,
50 mM Tris base, 2.1 mM EDTA, 50 mM NaCl, 5
mM DTT, and 0.2 mg/ml sonicated calf thymus DNA. The binding
of HisOBP and ICP 8 to oriS was monitored in Tris-glycine buffered
6% polyacrylamide gels as described previously. The reaction mixture
contained 2.5 fmol of an end-labeled restriction fragment containing
oriS or a mutant version thereof in a total volume of 20 µl. The
buffer was 0.38 M glycine, 50 mM Tris base, 10 mM MgCl
, 2.1 mM EDTA, 3 mM DTT, and
0.02 mg/ml BSA(17) . HisOBP and ICP 8 were included as
described in the figure legends. Incubation was for 10 min on ice.
Electrophoresis was for 3 h at 100 V at +4 °C. The dried gels
were subjected to autoradiography in the presence of an intensifying
screen.OBP to single-stranded DNA was
analyzed in a similar gel retardation experiment. Here 3.1 fmol of T65
was mixed with 14-124 pmol of HisOBP in a total volume of 20
µl. The reaction buffer was the same as above, and the samples were
run on 6% polyacrylamide gels. The conditions for the electrophoresis
were the same as above.DNase I Footprinting
9.5 fmol of an EcoRI-SphI restriction fragment derived from the
plasmid pORI (17) was end labeled at the EcoRI site
using [-P]dTTP (3000 Ci/mmol). It was mixed
with 5.4 pmol of His
OBP and 9.2 pmol of ICP 8 as indicated in the
legend of Fig.7. The reaction mixture was 20 µl and
consisted of 3 mM MgCl, 100 mM NaCl, 50
mM MOPS, pH 7.4, 1 mM DTT, 0.1 mM EDTA, 10%
glycerol, and 0.02 mg/ml BSA. The samples were incubated 15 min on ice.
They were then kept at 37 °C for 3 min immediately before the
addition of 0.2 units of DNase I. The cleavage reaction was allowed to
proceed for 3 min at 37 °C. The reaction was terminated by the
addition of 5 µl of 0.25 M EDTA and 0.01 mg/ml sonicated
calf thymus DNA. The samples were extracted by phenol and precipitated
with ethanol and subsequently analyzed on 8% polyacrylamide sequencing
gels.
OBP, ICP 8, and oriS is independent of the phasing of the
binding sites. A radiolabeled restriction fragment containing oriS
derived from the pORI series of plasmids (see ``Materials and
Methods'') was mixed with a constant amount of HisOBP (4.3
nmol) and ICP 8 (4.2 nmol) as indicated in the figure. The length of
the spacer sequence separating boxes I and II of oriS varied as shown.
The autoradiograph shows free DNA (lane 1), two complexes of
different stoichiometries between HisOBP and oriS (lane
3), and a complex consisting of HisOBP, ICP8, and oriS (lanes 4-8).
OBP and ICP 8
Is Determined by the DNA Ligand-Boehmer and Lehman (10) originally discovered that a stable and specific complex
could be formed between the HSV-1 origin binding protein and ICP 8. We
have now looked into the problem of how the formation of this complex
is affected by two different ligands: the duplex oligonucleotide PE
17/18, also referred to as box I, corresponding to the high affinity
binding site for OBP in oriS, and T65, a single-stranded
oligonucleotide that will efficiently bind ICP 8.OBP and ICP 8, respectively, on Superose 12 indicated
molecular weights of 38,000 and 170,000 ( Fig.1and Table 1). The expected value for ICP 8 was 128,000 calculated on
the basis of amino acid sequence. Nevertheless, these values indicate
that HisOBP and ICP 8 exist as monomers in solution.
OBP, ICP 8, and DNA. HisOBP, ICP 8, box
I, and T65 were mixed in equimolar amounts as indicated in the graphs.
Chromatography of the samples was carried out on a calibrated column of
Superose 12 as described under ``Materials and Methods.'' The
elution profiles of HisOBP and ICP 8 were monitored by
SDS-polyacrylamide gel electrophoresis. The elution of box I and T65
was followed by liquid scintillation counting. The positions of the
free components as determined in separate experiments are indicated by arrows.
OBP and ICP 8 were mixed in equimolar proportions a complex was
formed that eluted with an apparent molecular weight of approximately
215,000 ( Fig.1and Table 1). This value is consistent
with the formation of a heterodimer with a stoichiometry of 1:1.OBP and the box I duplex oligonucleotide
were mixed at an equimolar ratio a complex, HisOBP/box I, eluted
at an apparent molecular weight of 92,000. Neither free DNA nor free
HisOBP was observed. However, if either DNA or HisOBP was
added at a slight excess, e.g. a molar ratio of 2:1, the
expected amounts of free DNA or HisOBP, as the case might be,
could also be observed on the chromatogram (results not shown). This
indicates that 1 molecule of box I can bind 1 molecule of HisOBP
under these conditions. A recent report suggests that a single high
affinity binding site from oriS may bind two C-terminal domains of
OBP(31) . The apparently contradictory results have been
obtained using different techniques, and further experiments are
clearly needed to resolve this issue.OBP-ICP 8 complex was
investigated in two experiments where either the box I duplex
oligonucleotide PE17/18 or T65 was added to the HisOBP-ICP 8
complex (Fig.1c). In the first case all three
components coeluted at a position corresponding to a molecular weight
of 350,000. Speculation about the composition of this complex on the
basis of its chromatographic properties might be hazardous, but it
seems likely that these components might form a highly assymmetric
complex with a stoichiometry of 1:1:1. In order to investigate the
stoichiometry of this complex, a titration experiment was performed.
The concentrations of ICP 8 and the box I duplex oligonucleotide were
kept constant in the sample mixture, 3 and 1 µM,
respectively. Increasing amounts of OBP were then added resulting
in molar ratios of OBP to duplex oligonucleotide ranging from 0.3
to 3. The samples were chromatographed on Superose 12. The amount of
the ternary complex, denoted I in the inset of Fig.2,
was determined as shown in Fig.2. Similarly, we measured the
amount of the free duplex box I oligonucleotide represented by the
second peak in the chromatogram of Fig.2. Our results from the
titration experiments indicate that maximal amounts of ternary
complexes are obtained at approximately an equimolar ratio of OBP
to duplex box I oligonucleotide. We believe that the slight deviation
from the theoretical value of 100% can be explained by a dissociation
of components in the complex during chromatography. In the second
experiment we examined the influence of the single-stranded
oligonucleotide T65 on the formation of a HisOBP-ICP 8 complex.
The striking observation was made that HisOBP was completely
displaced from the complex and the previously described ICP 8-T65
complex emerged (Fig.1d). We speculate that a
conformational change of ICP 8 induced by single-stranded DNA caused
this effect.
OBP, and a box I duplex oligonucleotide. ICP 8 and the
box I duplex oligonucleotide PE 17/18 were included in the sample
mixture at constant concentrations, 3 and 1 µM
respectively. Increasing amounts of HisOBP, 0.3, 0.6, 1, 2, and 3
µM, was added to this mixture. The sample mixtures were
incubated on ice for minutes before performing a chromatography on
Superose 12 as described under ``Materials and Methods.'' The inset shows the elution profile from an experiment where 1
µM HisOBP was used. The first peak corresponds to the
putative ternary complex. The second peak corresponds to free DNA and
possibly also some binary complex between HisOBP and DNA. The
total amount of DNA was defined as the sum of the radioactive
oligonucleotide in peaks I and II. The relative amount of complex was
calculated as the content of radioactive oligonucleotide in peak I
divided by this value. The dashed line corresponds to the
maximal fraction of complex formed under these experimental
conditions.
Binding of ICP 8 and His
To further address the stoichiometry of the complex formed
between ICP 8 and single-stranded DNA, we subjected this complex to
digestion with micrococcus nuclease. A single-stranded polynucleotide,
T300, was synthesized by terminal transferase in the presence of
[OBP to Single-stranded
DNA-P]dTTP. Complexes between ICP 8 and the
uniformly labeled single-stranded polynucleotide T300 polynucleotide
were then digested with micrococcus nuclease. The cleavage products
were subjected to electrophoresis on sequencing gels. The results
revealed a periodic pattern of protection. The repeating unit was
exactly 14 nucleotides (Fig.3). The single-stranded
oligonucleotide T65 used in the gel filtration experiments above could
therefore bind four ICP 8 monomers.
OBP and single-stranded DNA. An experiment
using the gel retardation technique shows that HisOBP can bind to
a single-stranded oligonucleotide at high concentrations (Fig.4).
OBP binds to single-stranded DNA.
HisOBP and a radiolabeled single-stranded oligonucleotide T65 were
mixed as indicated. The samples were analyzed on 6% polyacrylamide gels
in a Tris-glycine buffer as described under ``Materials and
Methods.'' The positions of the free oligonucleotide and the
complex between HisOBP and T65 in the resulting autoradiograph of
the dried gel are shown.
OBP and T65 we have noted that HisOBP binds to T65 with an
apparent K of 10
M. (
)The complexes formed under these
conditions have a stoichiometry of 1:1. However, when complexes between
HisOBP and T65 were examined in protection experiments using
micrococcus nuclease there was no indication of a protection resulting
in a repeating pattern. Furthermore, we were never able to isolate
complexes between HisOBP and T65 using chromatography on Superose
12 as described above. We interpret these results to mean that the
interaction between HisOBP and single-stranded DNA is weak and
non-cooperative.His
We have tried to define the stoichiometry of
the complexes formed between OBP and GSTOBP Do Not Form Heterodimers on a
Single Binding SiteOBP and a single binding site
corresponding to the box I duplex oligonucleotide. Initial experiments
using gel filtration on Superose 12 demonstrated that HisOBP and
GSTOBP both occurred as monomers in solution (results not shown).
The formation of complexes between these proteins and the box I duplex
oligonucleotide was then examined in gel retardation experiments.
HisOBP and GSTOBP were mixed with the box I duplex
oligonucleotide. For each protein only one complex with the duplex
oligonucleotide was observed (Fig.5). When the two proteins
were mixed prior to the addition of the box I duplex oligonucleotide
two species were detected. The mobilities of these complexes were
identical with those previously seen. There was no indication of the
formation of a new species corresponding to a heterodimer of
HisOBP and GSTOBP (Fig.5). Our observations are in
agreement with those made by Martin et al.(32) using
truncated versions of OBP translated in vitro.
OBP and
GSTOBP are not formed on a duplex oligonucleotide containing a
single binding site. A radiolabeled box I duplex oligonucleotide (PE
17/18) was mixed with HisOBP and GSTOBP as indicated. The
samples were analyzed on 7% polyacrylamide gels. Complex I contains
HisOBP and box I. Complex II consists of GSTOBP and box
I.
Binding of ICP 8 to oriS Can Be Mediated by
His
The results presented in the first part of this
investigation showed that the stability of the interaction between OBP
and ICP 8 is influenced by the presence of a DNA ligand. We have now
examined the possibility that OBP can recruite and deliver ICP 8 to
single-stranded DNA appearing at oriS during the process of initiation
of DNA replication.OBPOBP was somewhat unpredictable. It was
unstable under dilute conditions and formed aggregates or precipitated
at concentrations above 2 mg/ml (results not shown). The addition of
bovine serum albumin or ICP 8 tended to stabilize the protein and
improve its ability to interact with DNA (results not shown). In the
experiments described below, we have therefore always included BSA in
the reaction mixtures.OBP
sustained the formation of one unique complex with a box I duplex
oligonucleotide. The HSV-1 origins of replication contains two good
binding sites for OBP, boxes I and II. An altered form of oriS has been
made. In this case the affinity of box I for OBP was reduced by two
orders of magnitude by the introduction of two point mutations in oriS
creating oriS(boxI mut)(13) . When a restriction fragment
containing oriS(boxI mut) was used in gel retardation experiments it
was evident that there was only one complex formed with HisOBP (Fig.6, lane 3). If ICP 8 alone was added to this
restriction fragment we could not detect the formation of a stable
complex ( Fig.6lane 2). However, when ICP 8 and
HisOBP were included in the same reaction mixture we now could
observe the formation of novel complexes (Fig.6, lanes
4-6). Strikingly, we could observe a gradual shift of the
HisOBP/oriS(boxI mut) complex toward a more slowly migrating
species. When ICP 8 was added in excess there was no further change in
the electrophoretic mobility of the complex (Fig.5, lane
6). The slowly migrating complex clearly contains ICP8,HisOBP
and oriS, but the stoichiometry is uncertain. We then looked at the
interaction between HisOBP, ICP 8, and restriction fragments
containing oriS where the distance between box I and box II had been
altered. We could also in this case observe that ICP 8 promoted the
formation of a slowly migrating complex. The mobilities of the
complexes containing oriS(boxI mut) or oriS(wt) obtained in the
presence of excess ICP 8 appeared to be identical (results not shown).
Furthermore, the formation of this species was independent of the
distance between box I and box II (Fig.7). We interpret these
results tentatively in the following way: ICP 8 and HisOBP bind
boxes I and II of oriS forming a complex in which two ICP8/HisOBP
heterodimers bind to 1 molecule of oriS (Fig.11a). The
interaction with box I and II seems to be distance independent and
non-cooperative. We also suggest that ICP 8 seems to further enhance
the affinity of HisOBP for DNA allowing the formation of the
slowly migrating complexes also in the presence of a mutated box I (Fig.6).
OBP mediates the binding of ICP 8
to oriS. A radiolabeled 100-base pair restriction fragment derived from
pORI(boxI mut), thus containing only one good binding site for OBP, was
mixed with HisOBP and ICP 8 as indicated. The samples were run on
6% polyacrylamide gels in a Tris-glycine buffer. The autoradiograph of
the dried gel shows free DNA (lane 1), a complex between
HisOBP and oriS (lane 2), and complexes containing
HisOBP, ICP 8, and box I (lanes
3-5).
OBP-ICP
8-oriS complex and a tentative complex between full-length OBP, ICP 8,
and oriS depicted as an intermediate in the initiation of DNA
synthesis. a, a model describing the interaction between
HisOBP, ICP 8, and oriS. HisOBP is represented by a lightly shaded circle. ICP 8 is darkly shaded. The
positions of boxes I-III are indicated. b, a tentative model
describing the activation of oriS. Two OBP dimers bind cooperatively to
oriS (Refs. 15, 17). The C-terminal domain of OBP is shown as a lightly shaded part of a circle and the N-terminal domain is
represented by the striped part of a circle. ICP 8, a darkly shaded circle, is brought to oriS through an
interaction with OBP. Two ICP 8 monomers are positioned at the AT-rich
spacer sequence and may detach from OBP as soon as this region becomes
single-stranded. Gradual ATP-dependent unwinding leads to a positioning
of ICP 8, aided by OBP, on single-stranded
DNA.
OBP and oriS. We then observed that the addition of the
single-stranded oligonucleotide T65 completely prevented ICP 8 from
binding to a complex between HisOBP and oriS (Fig.8).
OBP and oriS. The
reaction mixtures contained 1 fmol of a radiolabeled oriS containing
restriction fragment, 0.35 pmol HisOBP, 0.71 pmol ICP 8, and 0.24
pmol T65 as indicated in a total volume of 20 µl. Electrophoresis
was carried out as described for Fig.6.
His
It has previously been seen that OBP binds efficiently to
boxes I and II of oriS. Under certain conditions a weaker interaction
with box III can also be detected(15) . The AT-rich spacer
sequence, however, does not seem to interact with the protein. We have
now used the DNase I footprinting technique to investigate how ICP 8
interacts with oriS (Fig.9). Our results revealed that
HisOBP Positions ICP 8 at the AT-rich Spacer of
oriSOBP bound boxes I and II in oriS, but it did not affect the
cleavage pattern of the AT-rich sequence. ICP 8 alone did not produce
any footprint on oriS at all. In contrast, the simultaneous presence of
ICP 8 and HisOBP resulted not only in a footprint covering boxes I
and II, but it also gave a distinct protection of a part of the AT-rich
sequence ( Fig.9and 10). Notably, the phosphodiester bond of
nucleotide 43 of oriS was cleaved very poorly in the presence of ICP 8 (Fig.10). There was no change in the DNase I cleavage pattern
either upstream of box I or downstream of box II (Fig.9). These
observations indicate that boxes I and II are functionally assymmetric
and that ICP 8 is positioned in the immediate vicinity of the AT-rich
spacer sequence (Fig.11a).
OBP-ICP
8-oriS complex. A 100-base pair restriction fragment (9.5 fmol)
containing oriS from the plasmid pORI (see ``Materials and
Methods'') was end labeled at the EcoRI site. It was
mixed with HisOBP (5.4 pmol) and ICP 8 (9.2 pmol) as indicated.
DNase I footprinting was performed as described under ``Materials
and Methods.'' The positions of the binding sites for HisOBP,
boxes I, and II are shown. The arrow shows the position of a
phosphodiester bond uniquely protected in the HisOBP/ICP 8/oriS
complex (lane 4). The numbering of the nucleotides in oriS is
described in (17) .
OBP (Fig. 6, lane 3). d, oriS, HisOBP, and
ICP 8 (Fig.6, lane 4). The dotted line representing the scan in a is included as a
reference.
OBP and oris(boxI
mut) in the same way. We observed full protection of box II but a much
weaker footprint at box I. In addition, the phosphodiester bond at
position 49 in the AT-rich sequence was shielded from DNase I cleavage
(results not shown). In this experiment box II apparently served as the
strong binding site for HisOBP thereby delivering ICP 8 to a
position at the spacer sequence closer to box II.
OBP and ICP 8 were broken. We believe
that a conformational change of ICP 8 induced by single-stranded DNA
rendered this protein unable to bind to OBP. In this context it is of
interest to note that the intrinsic tryptophane fluorescence of ICP 8
is quenched substantially in the presence of single-stranded DNA
indicating that a conformational change might have taken
place(26) . Further experiments showed that a complex between
ICP 8 and single-stranded DNA produced a repeating structure in which
exactly 14 nucleotides were protected from nuclease cleavage by each
ICP 8 monomer. The results discussed above indicate that ICP 8 might
stimulate the helicase activity of OBP on model substrates by taking
part in a dynamic cycle. We may assume that ICP 8 can be actively
recruited to the replication fork by OBP during the first part of the
cycle. The interactions between OBP and ICP 8 will then be broken
allowing a precise positioning of ICP 8 on single-stranded DNA. In
other words, a controlled polymerization of ICP 8 on single-stranded
DNA can be expected to occur during the action of the helicase. It is,
however, not known if OBP has a role at the replication fork during the
infectious cycle. Possibly, the activity of OBP is restricted to a
local unwinding at the origin of replication. We therefore examined if
HisOBP could recruite ICP 8 to oriS. In the presence of ICP
8,HisOBP and oriS, a distinct slowly migrating complex was
observed in gel retardation experiments. DNase I footprinting
experiments further showed that ICP 8 was positioned close to the
AT-rich spacer sequence. A tentative model for this complex could be
suggested on the basis of the results presented in this article (Fig.11a). We first assume that a single HisOBP
monomer binds to each recognition sequence (this work, 32). It should
be noted that a recent report using a different technique suggests a
stoichiometry of 2:1 for this complex(31) . This interaction
might be stabilized specifically by ICP 8. Under these conditions two
ICP 8 monomers might be recruited to oriS and positioned at the AT-rich
spacer sequence (Fig.11a). The distance between the
two strong binding sites for OBP in oriS is sufficient to allow this to
take place. We have no indications that the conformation of the spacer
sequence is affected.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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