Role of the EBNA-1 protein in pausing of replication forks in the Epstein-Barr virus genome.

We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome. Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR. Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR. We conclude that a portion of the DNA binding domain is important for fork pausing. We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing. To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases. We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.

We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome. Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR. Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR. We conclude that a portion of the DNA binding domain is important for fork pausing. We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing. To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases. We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.
The Epstein-Barr virus latent origin of replication, oriP, was originally identified as a genetic element that conferred the ability to replicate autonomously upon small plasmids in EBV 1 -transformed cells (1). It was subsequently shown that oriP is composed of two essential elements separated by approximately 1 kb (2). One of these, the family of repeats (FR), contains 20 tandemly repeated copies of a 30-bp sequence, each containing a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. The FR is important for nuclear retention of the viral genome (3,4) and is also a transcriptional enhancer (5). The second essential region is termed the dyad symmetry (DS) region and contains a 65-bp sequence of dyad symmetry and four binding sites for EBNA-1. The EBNA-1 protein binds as a dimer to its sites in the FR and the DS region (6,7); moreover, EBNA-1 binds in a highly cooperative manner to the DS region (8,9). EBNA-1 binding to oriP induces bending of DNA resulting in distortion of the helical structure (10 -12). EBNA-1 is also involved in the formation of a looped DNA structure in which the FR and DS region are bound together through an EBNA-1 complex (13,14). The role of EBNA-1 in initiation of EBV replication and in the maintenance of the EBV genome still remains unknown.
Our laboratory has previously shown that replication of a plasmid (p174), containing oriP, initiates within or near the DS region and proceeds bidirectionally. Replication forks that proceed leftward are arrested within the FR; the other forks travel the remainder of the circular plasmid until they encounter the arrested forks at the FR. Thus, the plasmid p174 replicates predominantly in an asymmetric bidirectional manner in human cells since oriP contains both the sites for initiation and termination of replication (15). We have also analyzed replication intermediates from intact EBV genomes in four EBVpositive cell lines (721, 8866, Daudi, and Raji). We showed that replication initiates near or within the DS region as seen in the p174 plasmid; however, initiation events were also detected external to oriP. Replication forks also stall at the FR in all four EBV genomes, but the levels of termination at the FR vary among cell lines suggesting differences in the regulation of termination events in different EBV strains (16).
Since EBNA-1 binds to multiple sites within the FR, we have begun to investigate its role in the pausing of replication forks. Toward this aim, we have used an in vitro replication system in which replication of plasmids containing the FR is initiated from the SV40 origin in the presence of T antigen and a soluble HeLa cell extract. When EBNA-1 is present in these reactions, we found that accumulation of replication forks stalled at the FR is enhanced (17). We also found that reducing the number of the repeats from 20 to 6 still resulted in replication pausing, but pausing was not detected with two repeats. The substitution of three tandem copies of the DS region (12 EBNA-1 binding sites) for the FR also results in pausing of replication forks, suggesting that multiple EBNA-1 binding sites are important irrespective of spacer DNA sequences (18). It has recently been found that two cellular proteins from the BJAB cell line activated by 12-O-tetradecanoylphorbol-13-acetate can compete with EBNA-1 for binding to oriP (19,20). The possible role of these proteins in initiation of EBV replication or in the pausing of replication forks remains unknown.
Our previous studies used an EBNA-1 derivative that lacks a region with repeated glycine alanine residues (these have been shown not to be essential for replication). In this study, we show that the full-length EBNA-1 protein also enhances the stalling of replication forks at the FR in vitro. We also examined a series of truncated EBNA-1 proteins to map the amino acids that are important for pausing of replication forks. We show that the complete DNA binding and dimerization region of EBNA-1 is sufficient to elicit the pausing of replication forks and that deletion of a portion of the DNA binding domain abrogates the ability of EBNA-1 to elicit pausing. We have also tested the EBNA-1 protein for its ability to inhibit DNA unwinding by T antigen and dnaB, two replicative helicases. EBNA-1 can inhibit DNA unwinding by T antigen and dnaB in an orientation-independent manner.

MATERIALS AND METHODS
Protein Purification-Recombinant virus AcEBNA-1, which includes a BamHI and HindIII fragment containing the entire open reading frame of the EBNA-1 gene (B95-8 strain), was kindly provided by J. Hearing (11). EBNA-1 was purified from SF9 cells that were grown as monolayer cells in Grace's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Cells were infected with 10 plaque-forming units of AcEBNA-1 per cell, harvested 60 h postinfection, and purified in two steps by heparin-agarose and DNA affinity chromatography as described earlier (21). EBNA-1 lacking the glycinealanine repeat and EBNA451 were produced using a baculovirus expression system and purified to homogeneity as described previously (21,22). EBNA351, EBNA463-607, and EBNA470 -607 were produced in Escherichia coli and purified to homogeneity as described (8,23,24).
Nitrocellulose Filter Binding Assay for EBNA-1 Binding Activity-This assay was carried out as described previously (17,21).
Gel Retardation Assay-Gel-purified DNA fragments (SacI-XbaI 886-bp fragments containing 20 copies of the 30-bp EBV repeats were isolated from p1221 kindly provided by C. Tyree) were end labeled with [␣-32 P]dCTP using the Klenow fragment of DNA polymerase I. Different amounts of full-length EBNA-1 or EBNA-truncated mutants were prebound to 10 fmol of labeled DNA at room temperature for 10 min in 20 l of 300 mM NaCl, 10 mM MgCl 2 , 50 g/ml bovine serum albumin, and 5 g of salmon sperm DNA. Retardation was examined by electrophoresis on a 6% polyacrylamide gel at 170 V for 4 h. The gel was dried and exposed to XAR-5 (Kodak) film at Ϫ70°C with an intensifying screen for 24 h.
In Vitro DNA Replication Assay-The in vitro reaction was carried out as described previously (17). EBNA-1 was prebound to the DNA template (150 ng) at room temperature in a reaction mixture containing 50 mM HEPES-KOH (pH 7.5), 5 mM MgCl 2 , 0.5 mM dithiothreitol, 100 g/ml bovine serum albumin, 250 mM NaCl at room temperature for 10 min. 100 M each dATP, dGTP, and dTTP, 200 M each CTP, GTP, and UTP, [␣-32 P]dCTP (specific activity, 3 ϫ 10 3 cpm/pmol), 40 mM creatine phosphate, 40 mM phosphocreatine kinase, 0.5 g of T antigen, and 250 g of HeLa cytosol were then added, and reaction mixtures were incubated at 37°C for 1 h. Purified SV40 T antigen and HeLa cytosol, kindly provided by J. Boroweic and J. Hurwitz, were prepared as described previously (25). The amount of DNA synthesis was measured by the incorporation of [␣-32 P]dCTP into trichloracetic acid-insoluble material. The reactions were terminated by adjusting the reaction mixtures to 10 mM EDTA, 0.5% SDS, and 20 g/ml yeast tRNA. Proteins were digested with 100 g/ml proteinase K at 37°C for 1 h. Samples were extracted once with phenol-chloroform-isoamyl alcohol (25:24:1), and DNA was separated from unincorporated [␣-32 P]dCTP by Sephadex G-50 chromatography. DNA was precipitated with 2 volumes of cold ethanol.
Two-dimensional Gel Analysis of Replication Products-This analysis was carried out as described previously (17,26).
Preparation of the Substrates for Helicase Assay-EcoRI-SacI fragments from the p15neo or p6neo plasmids (plasmids provided by T. S. Chittenden and A. J. Levine) that contain 15 and 6 EBV repeats were cloned into to the EcoRI-SacI site of the M13mp18 and M13mp19 DNA, from which viral single-stranded circular DNAs M13mp18 -6FR, M13mp19 -6FR, M13mp18 -15FR, and M13mp19 -15FR were prepared. The substrates used for the helicase assay were prepared as described previously (27). A gel-purified DNA fragment (354-bp EcoRI-SacI fragment containing 6 repeats from p6neo plasmid) was annealed to the M13 mp18 -6FR or M13 mp19 -6FR single-stranded DNA at a 10:1 molar ratio of DNA fragment to either of the single-stranded M13 DNAs. The oligodeoxynucleotides were labeled with [␣-32 P]dATP when annealing was to the M13mp18 DNA and with [␣-32 P]dCTP for the M13mp19 DNA by Klenow polymerase; then, labeled DNA was separated from unincorporated [␣-32 P]dATP or dCTP by chromatography on Sepharose 4B.
Helicase Assay in the Presence of the EBNA-1-FR Complex-The T antigen helicase activity was measured as described previously (27,28). EBNA-1 was prebound to 10 fmol of the partial duplex DNA in a reaction mixture containing 100 mM potassium glutamate, 50 mM HEPES-KOH (pH 7.5), 5 mM MgCl 2 , 150 mM NaCl, and 100 g/ml bovine serum albumin. The reactions were incubated for 10 min at room temperature, and 60 mM ATP, 15 mM dithiothreitol, 1 g of creatine kinase, and 50 M creatine phosphate were added; the volume was then adjusted to 30 l. T antigen (0.3 g) was added, and reaction mixture was incubated for 30 min at 37°C. The reaction was stopped by the addition of 0.5% SDS, 20 mM EDTA, and 100 g/ml proteinase K. The reaction was incubated for 1 h at 37°C and analyzed by electrophoresis on a 6% polyacrylamide gel. The gel was dried and exposed for autoradiography at Ϫ70°C for 24 h. The dnaB helicase activity was performed as described previously (29).

Truncated Mutants of the EBNA-1 Protein Containing the Wild Type DNA Binding Domain Enhance the Pausing of Replication Forks in Vitro-
In this study, we used an in vitro replication system to examine the effect of full-length EBNA-1 protein and different truncated proteins on pausing of replication forks at the FR. Fig. 1 shows the different mutant EBNA-1 proteins used in this study and also summarizes the functional domains of EBNA-1 protein. The cell-free replication system initiates replication from the SV40 origin in the presence of a soluble HeLa cytosol and SV40 T antigen (25,38,39). The plasmid included in these reactions, pEco3Ј⌬, contains the SV40 origin of replication and the FR region of oriP. The FR was cloned so that the orientation of the FR relative to the SV40 origin is the same as the orientation of the FR relative to the DS region of oriP and will be referred to here as the in vivo orientation (a map of pEco3Ј⌬ is shown in Fig. 2).
The full-length EBNA-1 protein was expressed using the baculovirus system and purified using heparin-agarose and DNA affinity columns as described under "Materials and Methods." The EBNA-1 protein was prebound to the pEco3Ј⌬ plasmid in 300 mM NaCl and 10 mM MgCl 2 for 10 min at room temperature. Plasmid pEco3Ј⌬ DNA was incubated at 37°C for represent the glycine-alanine repeat, which has been shown not to be essential for EBNA-1 function (30,31). Several functional domains of the EBNA-1 protein (black rectangles) that have been elucidated are shown including: (i) separate core and flanking DNA binding domains (32,33), (ii) dimerization domain (32,33), (iii) domain responsible for the cooperative interaction between EBNA-1 dimers (8), (iv) loop formation domains (23,34,35), and (v) phosphorylated regions of the protein (36,37). Truncated EBNA-1 proteins utilized in these studies are shown at the bottom with the amino acids remaining in the proteins as indicated. 60 min (as described under "Materials and Methods") in a reaction mixture containing HeLa cytosol (as a source of replication proteins), T antigen, and [␣-32 P]dCTP to monitor DNA synthesis. The reaction mixture also included a source of energy in the form of ATP, creatine phosphate and phosphocreatine kinase, NTPs as substrate for the synthesis of RNA primers, and dNTPs. The reaction was stopped, and DNA was purified and digested with the restriction endonucleases XhoI and PvuI, resulting in two fragments of 2.3 and 4 kb (see Fig.  2). The two-dimensional gel electrophoresis technique (26) was used to analyze the resulting replication intermediates. As a control, reactions in the absence of EBNA-1 protein were performed; plasmid DNA was preincubated in the presence of 300 mM NaCl and 10 mM MgCl 2 for 10 min at room temperature, and in vitro replication was performed as described above.
The results of two-dimensional gel electrophoresis are shown in Fig. 3. In Fig. 3A, an autoradiogram of the control reaction (in the absence of EBNA-1) is shown (see Fig. 3G for a schematic representation of this autoradiogram). The 2.3-kb segment exhibits a pattern (simple Y arc) indicating that replication forks proceed through this segment from the SV40 replication origin located in the 4.0-kb segment. Replication forks proceed from the SV40 replication origin in a counterclockwise direction until they reach the FR, where some of these forks pause at two domains within the FR. The presence of a complete simple Y arc in the 2.3-kb fragment indicates that the impediment to replication fork movement is not very efficient and that most replication forks proceed through this site. Moreover, in the 4.0-kb fragment a diffuse triangular pattern is seen that corresponds to termination of replication across a broad region distinct from the FR. Hence, in the absence of the EBNA-1 protein, replication initiates from the SV40 origin, proceeds bidirectionally with approximately equal rates, and terminates at many sites in a region 180°opposite the SV40 origin.
A two-dimensional gel autoradiogram showing the replication intermediates produced in the presence of the full-length EBNA-1 is shown in Fig. 3B (see Fig. 3H for a schematic representation of this autoradiogram). Five criteria indicate the existence of a strong pause site near or in the FR. The first criterion is an increased accumulation of branched forks in the 2.3-kb fragment in the presence of EBNA-1 or EBNA-1 derivatives, in contrast to a slight accumulation in the absence of the EBNA-1 protein (compare Fig. 3 The second criterion is the absence of a complete Y arc for this 2.3-kb fragment, showing that the pause site is very efficient and that very few forks proceed through the pause site (Fig. 3, panels B-F). The third criterion is the diagonal spike emanating from the region in the gel where paused molecules are located. This indicates that termination is occurring specifically at or near the FR in the presence of EBNA-1. The fourth criterion is that the filled triangular region, resulting from random termination within the 4.0-kb segment, is either absent or greatly reduced (schematic representation on the Fig. 3, panels G and H), indicating that replication no longer terminates 180°opposite the SV40 origin. The last criterion used for the characterization of the pause site is the amount of completely replicated 2.3-kb fragment, depicted as a 2.3-kb black circle in Fig. 3, panels G and H. When pausing occurs in the presence of EBNA-1 protein or EBNA-1-truncated mutants, the amount of completely replicated 2.3-kb fragment is decreased relative to the amount observed in the control reaction that has been carried out without EBNA-1 protein (compare the intensity of the 2.3-kb spots in Fig. 3, panel A, with panels B-F). These five criteria indicate that, in the presence of EBNA-1, replication initiates at the SV40 origin and forks initially proceed bidirectionally, but the counterclockwise fork stalls at the FR. The other fork proceeds around the remainder of the plasmid until it converges upon the fork stalled at the FR, where termination occurs. Thus, in vitro replication switches from a bidirectional mode to asymmetric bidirection in the presence of full-length EBNA-1, similar to the mechanism we have observed for the p174 plasmid in vivo (15). We previously used an EBNA-1 derivative (21), which lacks a region with repeated glycine-alanine residues that have been shown not to be essential for replication (30,31). A comparison of the replication intermediates generated in the presence of the fulllength EBNA-1 (Fig. 3B) and EBNA-1 without the glycinealanine repeat (Fig. 3C) confirms that the presence of the glycine-alanine repeat is not essential for replication fork pausing in vitro.
We next examined replication intermediates synthesized in vitro in the presence of different truncated EBNA-1 proteins to determine the domains of the protein that contribute to the pausing of replication fork movement. Fig. 3D shows the twodimensional gel electrophoresis results of replication intermediates synthesized in the presence of the EBNA351 truncation mutant (see Fig. 1). EBNA351 was overproduced in E. coli and purified to greater than 90% purity as described previously (23). No differences in the replication intermediates were seen when the results for the EBNA351 and full-length EBNA-1 proteins were compared. In Fig. 3E, an autoradiogram of a two-dimensional gel after in vitro replication in the presence of EBNA451 is shown. This truncated EBNA containing amino acids 451-641 (see Fig. 1) was expressed using the baculovirus system and purified to near homogeneity (22). This protein retains the DNA binding and dimerization domains but cannot mediate looping between the FR and DS regions (22). A very strong signal indicating accumulation of the replication forks in the 2.3-kb fragment shows that EBNA451 enhances fork pausing near the FR (Fig. 3E). The results of two-dimensional gel electrophoresis of replication intermediates after in vitro replication in the presence of the EBNA459 -607 is shown in Fig. 3F. This protein contains the complete DNA binding and dimerization domains (8,33,40). EBNA459 -607 was overproduced in E. coli and purified to homogeneity as described previously (24). In our assay, with this EBNA-1 derivative, replication forks stall at levels similar to levels seen with the wild type protein. Thus, the C-terminal end of the EBNA-1 protein is not required for the pausing of replication forks at the FR. In The plasmid pEco3Ј⌬ (6.3 kb) is shown. The SV40 origin of replication (solid line) and the FR fragment (shaded rectangle) consisting of 20 tandem copies of a 30-bp sequence are indicated. A, the direction of replication in the absence of the EBNA-1 protein is shown by the arrows inside of the diagram. Replication initiates from the SV40 origin and proceeds bidirectionally, until termination occurs in the region approximately 180°opposite of the SV40 origin (17). B, the direction of replication in the presence of EBNA-1 protein is shown by the arrows inside of the diagram. Replication initiates from SV40 origin and proceeds bidirectionally. The counterclockwise fork traveling toward the repeat is arrested upon arrival at the repeat, while the clockwise fork traverses the circular plasmid to meet the arrested fork at or near the FR where termination occurs. Restriction endonuclease cleavage sites are indicated.
FIG. 3. EBNA-1 truncation fragments containing a non-mutated DNA binding domain produce a site at which replication forks pause when they proceed from the SV40 origin of replication. Strong pausing sites are observed when the pEco3Ј⌬ plasmid replicates in vitro in the presence of the full-length EBNA-1 proteins or different truncated derivatives of EBNA-1 protein that contain a non-mutated DNA binding domain. The DNA of pEco3Ј⌬ was preincubated in the absence (control) or in the presence of the full-length EBNA-1 protein or different truncated derivatives in 300 mM NaCl, 10 mM MgCl 2 for 10 min at room temperature. T antigen was added, and the reaction mixture was incubated for 60 min in the presence of [␣-32 P]dCTP. DNA was purified, cut with restriction endonucleases XhoI and PvuI, and analyzed by two-dimensional gel electrophoresis. The intense spots at the bottom right of each arc correspond to the position in the gel at which fully replicated linear segments of 2.3 and 4.0 kb migrate. A, faint sites where replication fork movement is paused are seen in control reaction mixtures lacking EBNA-1 protein. The 2.3-kb fragment contains two distinct spots that correspond to pause sites as indicated in panel G. Replication forks can progress through these pause sites. The strong triangular pattern represents double Y-shaped replication molecules produced from termination at multiple sites within the region approximately 180°opposite of the SV40 origin (panel G). Panels B-F, autoradiogram of the two-dimensional gel after in vitro reactions carried out in the presence of wild type EBNA-1 protein (B) and different truncated derivatives of EBNA-1 protein (panels C-F) (for the map of the truncated derivatives of EBNA-1, see Fig. 1). The 2.3-kb fragment generated a strong signal in every case, corresponding to the accumulation of replication forks near the FR (see panel H). Fewer replication forks are seen to pass through the pause sites when reactions were carried out in the presence of EBNA-1 derivatives than in their absence. Termination in the region opposite the SV40 origin was not detected. A site-specific termination signal is located in the 2.3-kb fragment indicated (panel H) (see text for explanation). C, autoradiogram of the two-dimensional gel of an in vitro reaction in the presence of EBNA-1 protein in which glycine-alanine repeat is deleted (21). D, autoradiogram of the two-dimensional gel of the replication intermediates (DNA was replicated in the presence of the EBNA351 protein (22)). E, autoradiogram of the two-dimensional gel of replication intermediates. DNA was replicated in the presence of the EBNA451 (22). F, the in vitro replication reaction was carried out in the presence of EBNA459 -607 protein (8,24). G and H, diagram of two-dimensional gel patterns generated from replicating molecules. Three classes of replication intermediates are diagrammed: segments containing a single replication fork generated from an external initiation site and summary, all EBNA-1 proteins examined that retain the DNA binding and dimerization domain can cause replication forks to pause at the FR. We conclude that amino acids from 1 to 458 and from 608 to 641 are not required to produce pausing of the replication machinery near the FR fragment mediated by the EBNA-1 protein. Our studies have not determined whether the region of EBNA-1 from 1 to 458 may play a role in modulating the ability of the EBNA-1 protein to produce pausing of replication forks.
The two-dimensional gel autoradiograms in Fig. 3, B and E, show that initiation also occurs within the 4.0-kb fragment of the pEco 3Ј⌬ plasmid (see linear map on Fig. 3, panels G and  H). This initiation is distinct from SV40 origin of replication and much less efficient compared with initiation from the SV40 origin. In these studies we have not addressed the question of whether or not the presence of the EBNA-1 protein in the reaction increases the efficiency of initiation within the 4.0-kb fragment.

EBNA Mutants Containing a Truncated DNA Binding Domain Do Not Result in Pausing of Replication Forks at the FR in Vitro-
The EBNA463-607 truncation mutant was next examined in the in vitro replication assay for its ability to enhance pausing of replication forks at the FR. EBNA463-607 was expressed in E. coli and purified to homogeneity as described previously (8). EBNA463-607 lacks four amino acids from the amino-terminal portion of the DNA binding domain. This protein has 2-fold lower affinity for sites of the DS compared with EBNA459 -607 (8). For the in vitro replication reactions we used a 200-fold (Fig. 4A) and a 400-fold (Fig. 4B) molar excess of this protein relative to the binding site to ensure complete saturation of each binding site. Under these conditions, a gel retardation assay (Fig. 5) indicated that each EBNA binding site of the FR contained a bound dimer of EBNA463-607. An autoradiogram of replication intermediates synthesized in the presence of a 200-fold molar excess of this protein showed that replication forks did not pause at the FR (Fig. 4A) and were, in fact, not detectably different from reactions in which EBNA-1 protein was not added. When replication was carried out in the presence of a 400-fold molar excess of EBNA463-607, pausing of replication forks at the FR was detected at a low level. The pausing of the replication forks in the presence of EBNA463-607, even in the presence of a 400fold molar excess of the protein, did not differ significantly from several of the control reactions, where no EBNA protein was added, carried out during the course of these studies.
We next analyzed EBNA470 -607, which is deleted for a portion of the DNA binding and dimerization domains. This EBNA-1 derivative was still able to bind DNA, but its affinity was reduced relative to wild type (8). EBNA470 -607 bound specifically to the FR region and to the sites 1 and 4 of the DS region, but it was severely impaired in its ability to bind the low affinity sites 2 and 3 of the DS element (8). We added a 1000-fold excess of EBNA470 -607 to the in vitro replication reactions. A gel retardation assay indicated (Fig. 5) that all 20 binding sites in the FR were occupied by EBNA470 -607 dimers. As shown in Fig. 4C in the presence of this EBNA mutant enhancement of replication fork pausing at the FR was not detected in in vitro replication reactions. The two-dimensional gel pattern is very similar to that obtained in the absence of the EBNA-1 protein; there was no accumulation of replication forks in the small 2.3-kb fragment, and termination occured 180°opposite the SV40 origin. Our two-dimensional gel results indicate that EBNA470 -607 does not enhance the stalling of replication forks at the FR.

EBNA-1 Inhibits the Unwinding Reaction Catalyzed by T Antigen and dnaB Helicases-
The helicase assay measures displacement (unwinding) of a DNA fragment that has been annealed onto single-stranded circular phage DNA. We therefore used this assay to determine whether an inhibition of helicase activity by EBNA-1 may play a role in the pausing of replication forks. The partial duplex DNA substrate containing EBNA-1 binding sites was constructed as described under "Materials and Methods." DNA fragments containing 6 or 15 EBNA-1 binding sites were cloned into M13mp18 and M13mp19 DNA. Single-stranded molecules from these constructs contained EBNA-1 binding sites in both orientations relative to the direction of helicase movement. The schematic representation of a substrate for the helicase assay is shown in Fig. 6A.
Two helicases were examined: T antigen of SV40, which translocates in the 3Ј to 5Ј direction (41)(42)(43), and dnaB protein of E. coli, which translocates in the 5Ј to 3Ј direction (see Fig.  7A) (29). Full-length EBNA-1 protein was prebound to the partial duplex DNA for 10 min at ambient temperature. T antigen, ATP, and an ATP regenerating system were then added, and the reaction was carried out for 60 min at 37°C. proceeding through the fragment (simple Y arc), segments in which two opposing replication forks converge at random locations within the fragment, which forms a filled triangular region (random termination), and segments in which replication forks pause at a site indicated by a vertical line. Nascent   (8,24) in 300 mM NaCl for 10 min at room temperature, and the in vitro reaction was done as described under "Materials and Methods." The DNA was purified, digested with XhoI and PvuI, and analyzed by two-dimensional gel electrophoresis. A faint replication fork pause site is seen in the 2.3-kb fragment that contains the FR. Two distinct spots are seen similar to the case when the reaction was carried out in the absence of the EBNA-1 protein as indicated in Fig. 3G. Y arc replication intermediates that progress past the pause site are indicated (see Fig. 3G). The diffuse triangular pattern indicates that termination occurs in the broad region opposite the SV40 origin as indicated (Fig. 3G) DNA was purified, and the products were resolved by nondenaturing polyacrylamide gel electrophoresis. The effect of EBNA-1 on the unwinding of partial duplexes is shown in Fig.  6, panels B and C. EBNA-1 inhibits DNA unwinding by T antigen regardless of the orientation and number of EBNA-1 binding sites (constructs containing 6 and 15 EBNA-1 binding sites have been tested). The same partial duplex DNA molecules were then tested in helicase assays using the dnaB helicase (Fig. 7). DNA unwinding by dnaB was also found to be inhibited by EBNA-1. The autoradiograms shown in Figs. 6, panels B and C, and 7B show that EBNA-1 inhibits the release of single-stranded DNA from partial duplexes independent of the orientation of the FR fragment and of the number of EBNA-1 binding sites, indicating that EBNA-1 inhibits helicase activity of SV40 T antigen and E. coli dnaB. DISCUSSION There are several similarities between pausing of replication forks at the FR and replication pause sites that have been observed in prokaryotes. Both types of pausing have been shown to be associated with a protein that binds specifically to a DNA sequence. Also, it has been shown that DNA helicase activity is inhibited at these sites. One difference between the termination pause sites in prokaryotes and the FR of the EBV site is that the pause sites in prokaryotes are polar and relatively efficient, whereas EBNA-1-mediated pausing at the FR occurs in both directions and is less efficient.
Termination of replication in the E. coli chromosome is confined to a zone approximately 180°opposite the origin. A mechanism has evolved to ensure that replication terminates specifically in this region by the strategic location of replication pause sites (for review, see Ref. 44). Two elements are involved in this mechanism: multiple DNA sequences of approximately 20 bp, termed Ter sites, and the Tus protein, which is a DNA binding protein that recognizes and binds specifically to the Ter sequences. The Ter sequences are present in six copies spread over a large region that generates a replication fork trap, i.e. replication forks can enter the termination zone but rarely exit from this region (45). Ter sequences do not contain a dyad symmetry region, indicating that the Tus protein probably binds as a monomer. The absence of a dyad symmetry region and monomeric binding of the Tus protein to the Ter sites may be responsible for the replication fork pausing in only one orientation with respect to the origin (28,46). In Bacillus subtilis, the replication arrest system consists of an inverted repeat (IR) region containing two inverted repeats (IR I and IR II) and the replication terminator protein (RTP), which associates as a dimer with IR I and IR II. The IR sequences contain core and auxiliary elements, each of which binds an RTP dimer and results in a polar barrier to replication fork movement (47,48). Pausing of replication forks in vitro occurs only when RTP binds to the core and auxiliary elements cooperatively (49 -51). The terminus regions of B. subtilis and E. coli share no apparent homology, nor do the RTP and Tus proteins. The mechanisms involved in replication fork pausing that are utilized in these organisms have not been fully elucidated and may, in fact, be different. Nevertheless, it has been shown that both proteins inhibit DNA unwinding by replicative helicases in vitro in an orientation-dependent manner (28,(52)(53)(54). In both cases, pausing is not absolute, and replication forks are eventually able to proceed through the terminator sites. Moreover, it has been shown recently that RTP and Tus proteins have the ability to block transcription in a polar manner (55). The passage of an RNA transcript can inactivate the replication termination site (55).
Several sites at which replication forks stall have been observed in eukaryotic systems. Thus far, however, a protein that is responsible for this effect has not been identified. Replication forks pause in the direction opposite to transcription at the 3Ј-ends of the rRNA transcription units for every organism that has been studied including human (56), murine, 2 Xenopus borealis, Xenopus laevis (57), and Saccharomyces cerevisiae (58). In yeast, it has been shown that transcription itself is not responsible for the pausing of replication forks in this region (59), but the possibility remains that some protein(s) from the transcription machinery are bound to this region and result in pausing of replication forks. The other pause sites that are responsible for replication fork stalling in yeast have been mapped to the tRNA genes of S. cerevisiae in vivo (60). These sites exhibit polarity and are similar to the pause sites of rDNA genes described earlier.
They are able to stall replication fork movement when replication machinery moves in the direction opposite to transcription. However, for tRNA pause sites, it has been shown that transcription per se is required for pausing of replication forks to occur (60).
One type of replication fork pause site in yeast that has been well characterized is a transient arrest of replication forks that occurs at the centromeres of chromosomes I, III, and IV (61). Replication fork movement pauses at the centromeres when approached from either direction; the efficiency of fork arrest is similar between chromosomes and plasmids. The pausing is clearly a result of the interaction between the centromere binding protein and centromeric DNA, since point mutations that abolish binding of centromere binding protein abolish replication fork pausing (61).
In this study, we have shown that replication forks pause at or near the FR in the presence of the EBNA-1 protein. Pausing can also occur in the absence of EBNA-1 but to a significantly lower degree. We consider two possible mechanisms that may be involved in pausing of replication forks in this region in the absence of EBNA-1. Replication forks may pause at the FR sequence because of some unusual secondary structure in the absence of bound protein. Alternatively, a protein or proteins present in the HeLa cytosol may bind to the FR fragment and result in pausing. It has been shown that three cellular proteins can bind to the FR (19,20,62). It is clear that pausing at the FR in the absence of the EBNA-1 protein is inefficient, and forks eventually proceed through the region.
A different mechanism for pausing at the FR is probably involved when EBNA-1 protein is present. In the presence of the FR-EBNA-1 complex, it is clear that relatively few forks can proceed through the pause site, although pausing is not absolute since some termination occurs in the region opposite the SV40 origin. It is possible that the interaction of EBNA-1 with one or more components of the replication machinery may elicit stalling of forks. Indeed, it has been shown that EBNA-1 is able to interact with DNA pol ␣-primase and human SSB. 3,4 However, EBNA459 -607 does not appear to interact with pol ␣-primase or human SSB, 3 even though it can inhibit replication fork movement.
It has been shown previously that EBNA-1 produced in the baculovirus expression system is a phosphoprotein that is phosphorylated on several serine residues (21). It has also been shown that phosphorylation of EBNA-1 occurs in vivo (36). The role of the phosphorylation of the EBNA-1 protein is unknown. It has been suggested that the phosphorylation on serines may exert a negative control over EBNA-1 function. 5 Here, we have shown that phosphorylation is not required for EBNA-1 or EBNA-1 derivatives to produce a pause site for replication fork movement. Using the in vitro replication reaction we examined EBNA-1 and EBNA-1 derivatives that have been produced using a baculovirus expression system and were presumably phosphorylated (21). We also assayed EBNA-1 derivatives that FIG. 6. Effect of EBNA-1 protein on the DNA unwinding activity of the T antigen. A, a schematic representation of helicase assay substrates M13mp18 and M13mp19 is shown. An EBV FR DNA fragment containing either 6 (6FR) or 15 (15FR) repeats was cloned in both orientations into M13mp18 and M13mp19 at the EcoRI and SacI sites; the orientation of the FR fragments is indicated by an arrow. The duplex region contains 354 bp for 6 repeats and 624 bp for 15 repeats. The direction of T antigen movement is indicated by an arrow and corresponds to T antigen translocation in the 3Ј-5Ј direction. B, polyacrylamide gel analysis of single-stranded DNA released by T antigen from M13mp18 -6FR and M13mp19 -6FR partial duplexes. 10 fmol of the M13mp18 -6 FR or M13 mp19 -6 FR partial duplex DNA was incubated with different amounts of EBNA-1 protein for 10 min at room temperature. T antigen (0.5 g) was added, and reactions were incubated at 37°C for 1 h. Lane 1, partial DNA duplex M13mp18 -6FR was boiled for 3 min. Lane 2, 10 fmol of partial duplex DNA, in a total volume of 30 l. Lane 3, EBNA-1 was not added to the reaction. Lanes 4 -6, reaction with 0.24, 0.12, and 0.06 g of EBNA-1 protein. In lane 7, the DNA M13mp19 -6FR was boiled for 3 min. In lane 8, only M13mp19 -6FR partial duplex (10 fmol) was present. In lane 9, EBNA-1 was omitted from the reaction. In lanes 10 -12, 0.24, 0.12, and 0.06 g of EBNA-1 protein were added to the reactions. C, polyacrylamide gel analysis of single-stranded DNA released by T antigen from M13mp18 -15FR and M13mp19 -15FR. 10 fmol of the substrate was incubated with different amounts of EBNA-1 protein for 10 min at room temperature. 0.5 g of T antigen was then added, and reaction mixtures were incubated at 37°C for 1 h. Lanes 1-6, M13mp18 -15FR partial duplex was examined in the following reactions: lane 1, the DNA substrate M13mp18 -15FR was boiled for 3 min; lane 2, 10 fmol of M13mp18 -15FR DNA substrate, as a control; lanes 3 and 4, the reaction was carried out in the absence of EBNA-1; lanes 5 and 6, reaction with 0.24 and 0.12 g of EBNA-1. In lanes 7-13, M13mp19 -15 FR partial duplex was used as a substrate in the following reactions: lane 7, M13mp19 -15FR was boiled for 3 min; lane 8, 10 fmol of DNA substrate as a control; lane 9, the reaction was carried out in the absence of EBNA-1 protein; lanes 10 -13, the reaction was carried out with 0.24, 0.12, 0.06, and 0.03 g of EBNA-1.
have been produced in E. coli and were not phosphorylated (see "Materials and Methods"). The fact that we did not detect any differences between the phosphorylated and non-phosphorylated EBNA-1 derivatives in the ability to stall replication forks movement suggests that the phosphorylation is not important for the replication fork pausing induced by EBNA-1.
To begin to understand the mechanism by which the EBNA-1-FR complex arrests replication fork movement, we examined the ability of the EBNA-1 protein bound to the FR site to inhibit helicase activity. In our assays, we used two different helicases, T antigen and dnaB. Both helicases were inhibited by the EBNA-1 protein bound to the FR (Figs. 6 and 7). When additional copies of the FR were added in trans, the inhibition of helicase activity was not detected (data not shown). This observation provides strong evidence that the mechanism by which EBNA-1 inhibits helicase activity requires EBNA-1 binding to its recognition sites in our helicase substrates. It is possible that the binding of the EBNA-1 protein to DNA is sufficient to inhibit helicase movement and that there is no protein-protein interaction between EBNA-1 and the helicase, as was shown for the Lac repressor (63). Lac repressor, when bound to its DNA binding site, can inhibit some, but not all helicases (63). However, a preliminary study suggests that the inhibition of helicase movement alone is not sufficient to result in pausing of replication fork movement at the FR. For example, replication fork pausing was not detected in vitro at the FR in the presence of EBNA470 -607, although helicase activity was inhibited in assays similar to those shown in Fig. 6. Experiments are in progress to determine whether pausing of replication forks at the FR is related to the ability of EBNA protein to inhibit helicase activity.
OriP has several features that are similar to some bacterial plasmid origins such as replication protein-mediated looping between short repeated sequences and a defined replication origin. These features may control EBV copy number, as has been proposed for some bacterial plasmids (for reviews see Refs. 64 -66). It has been shown that the EBNA-1 protein can mediate the formation of a loop in the DNA of the EBV oriP region in vitro. EBNA-1 binds to both the DS region and the FR, and the resulting EBNA-1 complexes interact resulting in the looping out of the intervening 1 kb of DNA (13,14). Here, using an in vitro replication reaction, we assayed the plasmid pEco3Ј⌬ that contains the FR but not the DS region. Our results indicate that the formation of a loop between the FR and DS region is not required for the pausing of replication forks that emanate from the SV40 origin of replication.
Another type of interaction between EBNA-1 molecules is termed cooperative binding, in which the binding of an EBNA-1 molecule at one site in the DS element positively affects the binding of EBNA-1 at its adjacent site (8,9). The cooperative binding interaction is mediated by EBNA-1 sequences between amino acids 470 and 607 (8,32) and therefore all of the EBNA-1 proteins used in this study can interact cooperatively. We have not yet determined whether cooperative interactions between EBNA-1 dimers on adjacent binding sites in the FR element are required to cause replication fork pausing.
We have shown in the present study that the portion of the EBNA-1 DNA binding region between amino acids 459 and 470 affects the ability of EBNA-1 to cause replication forks to pause in vitro. It has previously been shown, using a series of in vitro translated EBNA mutants, that the region between Gly-462 and Lys-477 contains residues important for DNA recognition (67). DNA binding activity is extremely sensitive to mutations in this region, and a dimer form of a synthetic peptide containing EBNA-1 amino acids 458 -478 is capable of binding DNA, but only nonspecifically (40). The crystal structure of the EBNA-1 DNA binding and dimerization domains bound to DNA has revealed that amino acids 461-470 form an extended chain that makes extensive contacts with the minor groove of the EBNA-1 recognition site (33). Loss of these contacts (as occurs in EBNA470 -607) results in decreased affinity for EBNA-1 recognition sites and likely in decreased DNA binding stability (7,8). The loss of the ability to cause replication forks to pause, which accompanied the deletion of amino acids 459 -469, suggests either that replication fork movement is blocked by the EBNA-1 extended chain in the minor groove of the DNA or that the resulting reduction in affinity and/or stability of EBNA-1 on DNA enables the passage of replication forks.
It is intriguing that the replication pause sites we have detected are all localized within a 2-3-kb region (although we have not screened the entire EBV genome for replication fork pause sites) (15)(16)(17). This may reflect the need for controlling the rate of replication in this region of the genome. It may also ensure that termination of replication occurs predominantly in this region because this region has the potential role of contributing to the fidelity of partitioning the EBV genome once replication has been completed. Furthermore, the proximity of these sites to oriP suggests that fork pausing may be involved in preventing the progression of forks proceeding from origins of replication that have reinitiated. This would indicate that pausing of forks may have a role in limiting duplication of the viral genome to once per cell cycle. A more detailed understanding of the mechanism underlying replication fork pausing will FIG. 7. Effect of EBNA-1 protein on the DNA unwinding activity of dnaB. A, schematic representation of the substrates that were used for the assay is shown. The direction of the FR fragment that was cloned into the EcoRI and SacI sites is indicated by the arrows. The direction of the translocation of dnaB along the partial duplex is shown by an arrow and corresponds to the 5Ј-3Ј direction of dnaB movement. B, polyacrylamide gel analysis of single-stranded DNA released by dnaB from M13mp18 -15FR or M13mp19 -15FR. 10 fmol of the substrate was incubated with different amounts of EBNA-1 protein for 10 min at room temperature, and then 2 mM ATP and dnaB was added to the reaction; the reaction was then carried out for 30 min at 37°C. In lanes 1-6, M13mp18 -15FR was used as a substrate for the following reactions: lane 1, the DNA substrate M13mp18 -15FR was boiled for 3 min; lane 2, reaction was carried out in the absence of EBNA-1; lanes 3-5, reaction with 0.24, 0.12, and 0.06 g of EBNA-1; lane 6, no EBNA-1, and no dnaB was added to the reaction. In lanes 7-12, M13mp19 -15 FR partial duplex was used in the following reactions; lane 7, the reaction was carried out in the absence of EBNA-1 protein; lanes 8 -10, reaction was carried out with 0.24, 0.12, and 0.06 g of EBNA-1; lane 11, M13mp19 -15FR substrate; lane 12, M13mp19 -15FR DNA was boiled for 3 min. allow us to understand how evolutionary mechanisms have adapted these critical elements in prokaryotes to the regulation of DNA replication in eukaryotes.