(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,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.
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 ()origin binding protein, OBP(5) .
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 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) .
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 GSTOBP and subsequent purification was performed essentially as
described(30) .
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
HisOBP 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.
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 HisOBP 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 His
OBP 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.
Figure 7:
The formation of complexes bewteen
HisOBP, 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 His
OBP (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 His
OBP and oriS (lane
3), and a complex consisting of His
OBP, ICP8, and oriS (lanes 4-8).
The Stability of Complexes between HisOBP 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.
Chromatography of
free HisOBP 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 His
OBP and ICP 8 exist as monomers in solution.
Figure 1:
Chromatography on Superose 12 of
complexes between HisOBP, ICP 8, and DNA. His
OBP, 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 His
OBP 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.
When
HisOBP 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.
In
an experiment where HisOBP and the box I duplex oligonucleotide
were mixed at an equimolar ratio a complex, His
OBP/box I, eluted
at an apparent molecular weight of 92,000. Neither free DNA nor free
His
OBP was observed. However, if either DNA or His
OBP was
added at a slight excess, e.g. a molar ratio of 2:1, the
expected amounts of free DNA or His
OBP, 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 His
OBP
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.
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 HisOBP-ICP 8 complex was
investigated in two experiments where either the box I duplex
oligonucleotide PE17/18 or T65 was added to the His
OBP-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 His
OBP-ICP 8 complex.
The striking observation was made that His
OBP 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.
Figure 2:
The formation of a ternary complex between
ICP 8, HisOBP, 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 His
OBP, 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 His
OBP 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 His
OBP 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.
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 HisOBP and single-stranded DNA. An experiment
using the gel retardation technique shows that His
OBP can bind to
a single-stranded oligonucleotide at high concentrations (Fig.4).
Figure 4:
HisOBP binds to single-stranded DNA.
His
OBP 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 His
OBP and T65 in the resulting autoradiograph of
the dried gel are shown.
Furthermore, in experiments employing quenching of
tryptophane fluorescens as a method of detecting an interaction between
HisOBP and T65 we have noted that His
OBP 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
His
OBP 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 His
OBP and T65 using chromatography on Superose
12 as described above. We interpret these results to mean that the
interaction between His
OBP and single-stranded DNA is weak and
non-cooperative.
Figure 5:
Heterodimers between HisOBP and
GST
OBP are not formed on a duplex oligonucleotide containing a
single binding site. A radiolabeled box I duplex oligonucleotide (PE
17/18) was mixed with His
OBP and GST
OBP as indicated. The
samples were analyzed on 7% polyacrylamide gels. Complex I contains
His
OBP and box I. Complex II consists of GST
OBP and box
I.
Early in this series of investigations we noted
that the behavior of HisOBP 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.
We have previously noted that HisOBP
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 His
OBP (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
His
OBP 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
His
OBP/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,His
OBP
and oriS, but the stoichiometry is uncertain. We then looked at the
interaction between His
OBP, 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 His
OBP bind
boxes I and II of oriS forming a complex in which two ICP8/His
OBP
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 His
OBP for DNA allowing the formation of the
slowly migrating complexes also in the presence of a mutated box I (Fig.6).
Figure 6:
HisOBP 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 His
OBP 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
His
OBP and oriS (lane 2), and complexes containing
His
OBP, ICP 8, and box I (lanes
3-5).
Figure 11:
Models describing the HisOBP-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
His
OBP, ICP 8, and oriS. His
OBP 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.
Finally, we wanted to examine the effect of
single-stranded DNA on the formation complexes between ICP
8,HisOBP and oriS. We then observed that the addition of the
single-stranded oligonucleotide T65 completely prevented ICP 8 from
binding to a complex between His
OBP and oriS (Fig.8).
Figure 8:
A single-stranded oligonucleotide prevents
ICP 8 from binding to a complex between HisOBP and oriS. The
reaction mixtures contained 1 fmol of a radiolabeled oriS containing
restriction fragment, 0.35 pmol His
OBP, 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.
Figure 9:
A DNase I footprint of a HisOBP-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 His
OBP (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 His
OBP,
boxes I, and II are shown. The arrow shows the position of a
phosphodiester bond uniquely protected in the His
OBP/ICP 8/oriS
complex (lane 4). The numbering of the nucleotides in oriS is
described in (17) .
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 HisOBP (Fig. 6, lane 3). d, oriS, His
OBP, and
ICP 8 (Fig.6, lane 4). The dotted line representing the scan in a is included as a
reference.
We also investigated the
structure of the complex formed between ICP 8,HisOBP 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 His
OBP thereby delivering ICP 8 to a
position at the spacer sequence closer to box II.
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 HisOBP 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
His
OBP could recruite ICP 8 to oriS. In the presence of ICP
8,His
OBP 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 His
OBP
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.
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.