©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The DNA Ligands Influence the Interactions between the Herpes Simplex Virus 1 Origin Binding Protein and the Single Strand DNA-binding Protein, ICP-8 (*)

(Received for publication, January 5, 1995; and in revised form, May 25, 1995)

Claes M. Gustafsson (§) Maria Falkenberg Stina Simonsson Hadi Valadi Per Elias (¶)

From theDepartment of Medical Biochemistry, University of Göteborg, Medicinaregatan 9, S-413 90 Göteborg, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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,DeltaOBP, 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 DeltaOBP, 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 DeltaOBP and ICP 8. We therefore suggest that the interaction between DeltaOBP 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.


INTRODUCTION

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 (^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 OBPbulletoriS 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.


MATERIALS AND METHODS

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 [alpha-P]dTTP 80 Ci/mmol. T300 was purified by chromatography on Bio-Gel P10 (Bio-Rad).

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) .

Purification of HisDeltaOBP, GSTDeltaOBP, and ICP 8

A pET3A expression vector for the C-terminal DNA-binding domain of OBP (DeltaOBP) was modified by the insertion of a duplex oligonucleotide introducing the amino acid sequence MetSerHis(6)GlyThrGly at the N terminus. The cells transformed with the pET3a/HisDeltaOBP 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.

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 GSTDeltaOBP 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 of ProteinbulletDNA Complexes

Complexes between HisDeltaOBP, 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.

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 HisDeltaOBP 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.

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 ProteinbulletDNA Complexes

The binding of HisDeltaOBP and GSTDeltaOBP 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 HisDeltaOBP and GSTDeltaOBP 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 HisDeltaOBP 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), 2.1 mM EDTA, 3 mM DTT, and 0.02 mg/ml BSA(17) . HisDeltaOBP 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.

The binding of HisDeltaOBP 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 HisDeltaOBP 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 [alpha-P]dTTP (3000 Ci/mmol). It was mixed with 5.4 pmol of HisDeltaOBP 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(2), 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.


Figure 7: The formation of complexes bewteen HisDeltaOBP, 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 HisDeltaOBP (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 HisDeltaOBP and oriS (lane 3), and a complex consisting of HisDeltaOBP, ICP8, and oriS (lanes 4-8).




RESULTS

The Stability of Complexes between HisDeltaOBP 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 HisDeltaOBP 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 HisDeltaOBP and ICP 8 exist as monomers in solution.


Figure 1: Chromatography on Superose 12 of complexes between HisDeltaOBP, ICP 8, and DNA. HisDeltaOBP, 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 HisDeltaOBP 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 HisDeltaOBP 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 HisDeltaOBP and the box I duplex oligonucleotide were mixed at an equimolar ratio a complex, HisDeltaOBP/box I, eluted at an apparent molecular weight of 92,000. Neither free DNA nor free HisDeltaOBP was observed. However, if either DNA or HisDeltaOBP was added at a slight excess, e.g. a molar ratio of 2:1, the expected amounts of free DNA or HisDeltaOBP, 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 HisDeltaOBP 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 HisDeltaOBP-ICP 8 complex was investigated in two experiments where either the box I duplex oligonucleotide PE17/18 or T65 was added to the HisDeltaOBP-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 DeltaOBP were then added resulting in molar ratios of DeltaOBP 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 DeltaOBP 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 HisDeltaOBP-ICP 8 complex. The striking observation was made that HisDeltaOBP 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, HisDeltaOBP, 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 HisDeltaOBP, 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 HisDeltaOBP 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 HisDeltaOBP 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 HisDeltaOBP to Single-stranded DNA

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 [alpha-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.


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 HisDeltaOBP and single-stranded DNA. An experiment using the gel retardation technique shows that HisDeltaOBP can bind to a single-stranded oligonucleotide at high concentrations (Fig.4).


Figure 4: HisDeltaOBP binds to single-stranded DNA. HisDeltaOBP 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 HisDeltaOBP 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 HisDeltaOBP and T65 we have noted that HisDeltaOBP binds to T65 with an apparent K of 10M. (^2)The complexes formed under these conditions have a stoichiometry of 1:1. However, when complexes between HisDeltaOBP 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 HisDeltaOBP and T65 using chromatography on Superose 12 as described above. We interpret these results to mean that the interaction between HisDeltaOBP and single-stranded DNA is weak and non-cooperative.

HisDeltaOBP and GSTDeltaOBP Do Not Form Heterodimers on a Single Binding Site

We have tried to define the stoichiometry of the complexes formed between DeltaOBP and a single binding site corresponding to the box I duplex oligonucleotide. Initial experiments using gel filtration on Superose 12 demonstrated that HisDeltaOBP and GSTDeltaOBP 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. HisDeltaOBP and GSTDeltaOBP 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 HisDeltaOBP and GSTDeltaOBP (Fig.5). Our observations are in agreement with those made by Martin et al.(32) using truncated versions of OBP translated in vitro.


Figure 5: Heterodimers between HisDeltaOBP and GSTDeltaOBP are not formed on a duplex oligonucleotide containing a single binding site. A radiolabeled box I duplex oligonucleotide (PE 17/18) was mixed with HisDeltaOBP and GSTDeltaOBP as indicated. The samples were analyzed on 7% polyacrylamide gels. Complex I contains HisDeltaOBP and box I. Complex II consists of GSTDeltaOBP and box I.



Binding of ICP 8 to oriS Can Be Mediated by HisDeltaOBP

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.

Early in this series of investigations we noted that the behavior of HisDeltaOBP 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 HisDeltaOBP 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 HisDeltaOBP (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 HisDeltaOBP 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 HisDeltaOBP/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,HisDeltaOBP and oriS, but the stoichiometry is uncertain. We then looked at the interaction between HisDeltaOBP, 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 HisDeltaOBP bind boxes I and II of oriS forming a complex in which two ICP8/HisDeltaOBP 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 HisDeltaOBP for DNA allowing the formation of the slowly migrating complexes also in the presence of a mutated box I (Fig.6).


Figure 6: HisDeltaOBP 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 HisDeltaOBP 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 HisDeltaOBP and oriS (lane 2), and complexes containing HisDeltaOBP, ICP 8, and box I (lanes 3-5).




Figure 11: Models describing the HisDeltaOBP-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 HisDeltaOBP, ICP 8, and oriS. HisDeltaOBP 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,HisDeltaOBP and oriS. We then observed that the addition of the single-stranded oligonucleotide T65 completely prevented ICP 8 from binding to a complex between HisDeltaOBP and oriS (Fig.8).


Figure 8: A single-stranded oligonucleotide prevents ICP 8 from binding to a complex between HisDeltaOBP and oriS. The reaction mixtures contained 1 fmol of a radiolabeled oriS containing restriction fragment, 0.35 pmol HisDeltaOBP, 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.



HisDeltaOBP Positions ICP 8 at the AT-rich Spacer of oriS

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 HisDeltaOBP 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 HisDeltaOBP 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).


Figure 9: A DNase I footprint of a HisDeltaOBP-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 HisDeltaOBP (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 HisDeltaOBP, boxes I, and II are shown. The arrow shows the position of a phosphodiester bond uniquely protected in the HisDeltaOBP/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 HisDeltaOBP (Fig. 6, lane 3). d, oriS, HisDeltaOBP, 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,HisDeltaOBP 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 HisDeltaOBP thereby delivering ICP 8 to a position at the spacer sequence closer to box II.


DISCUSSION

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 HisDeltaOBP 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 HisDeltaOBP could recruite ICP 8 to oriS. In the presence of ICP 8,HisDeltaOBP 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 HisDeltaOBP 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.


FOOTNOTES

*
This work was supported by Swedish Cancer Society Grant 2552-B94-08XAC, Medical Research Council Grant B 95-13Y-10442-03C, and the Assar Gabrielsson Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Support was given by the Göteborg Medical Society.

To whom correspondence should be addressed. Tel.: 46-31-773-3486; Fax: 46-31-416108; per.elias{at}medkem.gu.se.

^1
The abbreviations used are: HSV-1, herpes simplex virus type 1; OBP, origin binding protein; ICP 8, infected cell protein 8; BSA, bovine serum albumin; DTT, dithiothreitol.

^2
C. M. Gustafsson, unpublished observations.


REFERENCES

  1. Alberts, B. M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49,1-12
  2. Kornberg, A., and Baker, T. A. (1992) DNA Replication , 2nd Ed, Freeman, New York
  3. Stillman, B. (1994) Cell 78,725-728 [Medline] [Order article via Infotrieve]
  4. Skaliter, R., and Lehman, I. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,10665-10669 [Abstract/Free Full Text]
  5. Elias, P., O'Donnell, M. E., Mocarski, E. S., and Lehman, I. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,6322-6326 [Abstract]
  6. Olivio, P. D., Nelson, N. J., and Challberg, M. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,5414-5418 [Abstract]
  7. Bruckner, R. C, Crute, J. J., Dodson, M. S., and Lehman, I. R. (1991) J. Biol. Chem. 266,2669-2674 [Abstract/Free Full Text]
  8. Fierer, D. S., and Challberg, M. D. (1992) J. Virol. 66,3986-3995 [Abstract]
  9. Gorbalenya, A. E., and Koonin, E. V. (1993) Curr. Opinion Struct. Biol. 3,419-429
  10. Boehmer, P. E., and Lehman, I. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,8444-8448 [Abstract/Free Full Text]
  11. McLean, G. W., Abbotts, A. P., Parry, M. E., Marsden, H. S., and Stow, N. D. (1994) J. Gen. Virol. 75,2699-2706 [Abstract]
  12. Elias, P., and Lehman, I. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2959-2963 [Abstract]
  13. Elias, P., Gustafsson, C. M., Hammarsten, O. and Stow, N. D. (1990) J. Biol. Chem. 267,17424-17429 [Abstract/Free Full Text]
  14. Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 266,24621-24626 [Abstract/Free Full Text]
  15. Elias, P., Gustafsson, C. M., Hammarsten, O., and Stow, N. D. (1992) J. Biol. Chem. 267,17424-17429 [Abstract/Free Full Text]
  16. Hazuda, D. J., Perry, H. C., and McClements, W. L. (1992) J. Biol. Chem. 267,17424-17429 [Abstract/Free Full Text]
  17. Gustafsson, C. M., Hammarsten, O., Falkenberg, M. and Elias, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,4629-4633 [Abstract]
  18. Boehmer, P. E., Dodson, M. S., and Lehman, I. R. (1993) J. Biol. Chem. 268,1220-1225 [Abstract/Free Full Text]
  19. Boehmer, P. E., Craigie, M. C., Stow, N. D., and Lehman, I. R. (1994) J. Biol. Chem. 269,29329-29334 [Abstract/Free Full Text]
  20. O'Donnell, M. E., Elias, P., Funnell, B. E., and Lehman, I. R. (1987) J. Biol. Chem. 262,4260-4266 [Abstract/Free Full Text]
  21. Hernandez, T. R., and Lehman, I. R. (1990) J. Biol. Chem. 265,11227-11232 [Abstract/Free Full Text]
  22. Ruychean, W. T. (1983) J. Virol. 46,661-666 [Medline] [Order article via Infotrieve]
  23. Gao, M., Bouchey, J., Curtin, K., and Knipe, D. M. (1988) Virology 163,319-329 [CrossRef][Medline] [Order article via Infotrieve]
  24. Leinbach, S. S., and Heath, L. S. (1989) Biochim. Biophys. Acta 1008,281-286 [Medline] [Order article via Infotrieve]
  25. Wang, Y., and Hall, J. D. (1990) J. Virol. 64,2082-2089 [Medline] [Order article via Infotrieve]
  26. Ruychean, W. T., and Olson, J. W. (1992) J. Virol. 66,6273-6279 [Abstract]
  27. Gupte, S. S., Olson, J. W., and Ruychean, W. T. (1991) J. Biol. Chem. 266,11413-11416 [Abstract/Free Full Text]
  28. Dutch, R. E., and Lehman, I. R. (1993) J. Virol. 67,6945-6949 [Abstract]
  29. Bortner, C., Hernandez, T. R., Lehman, I. R., and Griffith, J. (1993) J. Mol. Biol. 231,241-250 [CrossRef][Medline] [Order article via Infotrieve]
  30. Martinez, R., and Edwards, C. A. (1993) Protein Exp. Purif. 4,32-37 [CrossRef][Medline] [Order article via Infotrieve]
  31. Fierer, D. S., and Challberg, M. D. (1995) J. Biol. Chem. 270,7330-7334 [Abstract/Free Full Text]
  32. Martin, D. W., Munoz, R. M., Oliver, D., Subler, M. A., and Deb, S. (1994) Virology 198,71-80 [CrossRef][Medline] [Order article via Infotrieve]
  33. Crute, J. J., Tsurumi, T., Zhu, L., Weller, S. K., Olivo, P. D., Challberg, M. D., Mocarski, E. S., and Lehman, I. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,2186-2189 [Abstract]
  34. Digard, P., and Coen, D. M. (1990) J. Biol. Chem. 265,17393-17396 [Abstract/Free Full Text]
  35. Digard, P., Bebrin, W. R., Weisshart, K., and Coen, D. M. (1993) J. Virol. 67,398-406 [Abstract]
  36. Tenney, D. J., Michelett, P. A., Stevens, J. T., Hamatake, R. K., Matthews, J. T., Sanchez, A. R., Hurlburt, W. W., Bifano, M., and Cordingly, M. G. (1993) J. Virol. 67,534-547
  37. Stow, N. D. (1993) Nucleic Acids Res. 21,87-92 [Abstract]
  38. Monahan, J., Barlam, T. F., Crumpacker, C. S., and Parris, D. S. (1993) J. Virol. 67,5922-5931 [Abstract]
  39. Lohman, T. M. (1993) J. Biol. Chem. 268,2269-2272 [Abstract/Free Full Text]
  40. Amarunga, M., and Lohman, T. (1993) Biochemistry 32,6815-6820 [Medline] [Order article via Infotrieve]
  41. Burke, R. L., Munn, M., Barry, J., and Alberts, B. M. (1985) J. Biol. Chem. 260,1711-1722 [Abstract]
  42. Dornreiter, I., Erdile, L. F., Gilbert, I. U., von Winkler, D., Kelly, T. J., and Fanning, E. (1992) EMBO J. 11,769-776 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.