©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleoprotein Complexes and DNA 5`-Ends at oriP of Epstein-Barr Virus (*)

Hans Helmut Niller (§) , Gerald Glaser , Ruth Knüchel (1), Hans Wolf

From the (1) Institut für Medizinische Mikrobiologie und Hygiene, Institut für Pathologie, Universität Regensburg, Franz-Josef-Strau-Allee 11, D-93053 Regensburg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Understanding protein-DNA interactions in vivo at origins of DNA replication throughout the cell cycle may shed further insight on the mechanisms of initiation and replication control. The Burkitt's lymphoma cell line Raji harbors multiple copies of latent Epstein-Barr virus. Once per cell cycle the origin of plasmid replication of Epstein-Barr virus provides replication function in cis for the viral DNA. Here we examined in vivo nucleoprotein complexes on the initiator element of the origin before and after DNA synthesis. For this purpose Raji cells were synchronously growth arrested in G phase by mimosine and in mitosis by colchicine, respectively. The association of the initiator element with proteins was visualized by footprinting with dimethyl sulfate and ligation mediated polymerase chain reaction. Methylation patterns indicated a novel binding activity within each element of a nonamer repeated three times at the initiator element. This activity was strongly diminished in mitotic cells. Furthermore, 5`-ends of Epstein-Barr virus DNA were mapped to the nonamers by ligation mediated polymerase chain reaction, suggesting potential initiation sites for replication from DS.


INTRODUCTION

Plasmid replication of Epstein-Barr virus (EBV)() may serve as a viral model system for chromosomal DNA replication in higher eukaryotes (DePamphilis, 1988). EBV persists in lymphoid cells, such as the Raji cell line derived from an African Burkitt's lymphoma, in a tightly latent state as a circular plasmid in multiple copies (Adams and Lindahl, 1975; Lindahl et al., 1976; Nonoyama and Pagano, 1972). Like cellular chromosomes, each viral plasmid is replicated once per cell cycle in the early synthesis (S) phase (Adams, 1987; Gussander and Adams, 1984; Hampar et al., 1974; Yates and Guan, 1991). The replication of EBV-derived plasmids is dependent on oriP, the viral protein EBNA1, and on a set of mostly unknown cellular proteins (Lupton and Levine, 1985; Reisman et al., 1985; Yates et al., 1984, 1985). OriP consists of two sequence elements, the family of repeat element (FR) and the dyad symmetry element (DS), separated by a stretch of about 960 base pairs of unique DNA dispensable for ori function (Lupton and Levine, 1985; Reisman et al., 1985). FR contains 20 times a 30-base pair repeat in tandem (Lupton and Levine, 1985; Reisman et al., 1985), each containing an EBNA1 binding site (Hsieh et al., 1993; Rawlins et al., 1985). In EBNA1 expressing primate cells, FR is essential for the stable maintenance of EBV plasmids (Lupton and Levine, 1985; Reisman et al., 1985; Wysokenski and Yates, 1989). The element contains the termination site for replication (Dhar and Schildkraut, 1991; Gahn and Schildkraut, 1989) and works as a replication enhancer (Wysokenski and Yates, 1989). Furthermore, it functions as a transcriptional enhancer (Reisman and Sugden, 1986; Wysokenski and Yates, 1989). DS contains four EBNA1 binding sites and a region of dyad symmetry encompassing EBNA1 binding sites four and three (Hsieh et al., 1993; Lupton and Levine, 1985; Rawlins et al., 1985; Reisman et al., 1985). In EBNA1 expressing primate cells DS most likely functions as the physical origin of bidirectional replication of EBV-derived plasmids (Gahn and Schildkraut, 1989; Platt et al., 1993; Wysokenski and Yates, 1989). Since initiation of DNA replication from oriP is a strictly regulated and dynamic event, we asked if there are variations in the proteins associated with the initiator element of oriP in vivo before and after S phase. Therefore, we decided to examine the state of in vivo protein-DNA interactions at the initiator element of oriP in synchronously before and after S phase growth-arrested Raji cells. Methylation protected and sensitive nucleotides were visualized using the technique of ligation mediated polymerase chain reaction (LMPCR) (Mueller and Wold, 1989). The exact physical initiation points or areas of EBV plasmid replication are not yet known at nucleotide resolution. Therefore, we decided to use the same method to visualize 5`-ends of viral DNA within the initiator element of oriP.


EXPERIMENTAL PROCEDURES

Tissue Culture

Raji cells were maintained in suspension cultures of RPMI 1640 medium containing 10% fetal calf serum, 2 mM glutamine, 50 units of penicillin/ml, and 50 µg of streptomycin/ml under 5% CO and 37 °C.

Flow Cytometric Analysis

For flow cytometric analysis cells were harvested, fixed with 70% methanol for at least 1 h, resuspended in 1 ml of phosphate-buffered saline (PBS) (Saluz and Jost, 1990), digested with 100 µg of RNase A for 1 h at 37 °C, stained with 50 µl of propidium iodide (1 mg/ml), and scanned on a Becton Dickinson FACScan analyzer using Cellfit software.

DMS in Vivo Footprinting

For each footprint 10 cells were harvested by centrifugation, washed with PBS (Saluz and Jost, 1990), resuspended in 1 ml of PBS, and incubated at room temperature for 5 min with 10 µl of dimethyl sulfate (DMS). The reaction was stopped by the addition of 5 ml of ice-cold DMS stop solution (1% of bovine serum albumin and 100 µM -mercaptoethanol dissolved in PBS) (Saluz and Jost, 1990). Cells were pelleted and washed once more with DMS stop solution and two more times with PBS. Cells were resuspended in 1 ml of PBS. Genomic DNAs were isolated according to standard methods (Saluz and Jost, 1990; Sambrook et al., 1989), either before sequencing reactions or after DMS treatment of cells. DMS-treated purified DNA was subjected to piperidine treatment (Maxam and Gilbert, 1980). For visualization by LMPCR, 2 µg of sequenced or footprinted DNA were analyzed according to the protocol of Garrity and Wold(1992) with some modifications. The following sets of oligonucleotides i, ii, and iii for the LMPCR assay of both strands were used, 5` to 3`: lower strand (i) 8927-GGTTCACTACCCTCGTGGAATCCTG-8951, (ii) 8931-CACTACCCTCGTGGAATCCTGACCC-8955, (iii) 8969-CCGTGACAGCTCATGGGGTGGGAGATATC-8997; upper strand (i) 9229-GGCTACACCAACGTCAATCAGAGGG-9205, (ii) 9205-GGCCTGTGTAGCTACCGATAAGCGG-9181, (iii) 9195-GCTACCGATAAGCGGACCCTCAAGAGG-9169. The first strand primer extension reaction was done in 10 mM KCl, 10 mM (NH)SO, 20 mM Tris-HCl, 2 mM MgSO, 0.1% Triton X-100, pH 8.8, 25 °C (Vent buffer, New England Biolabs), containing 0.3 pmol of primer i, 240 µM each dNTP, and 1 unit of Vent (exo-) DNA polymerase (New England Biolabs) for 5 min at 94 °C, 30 min at 60 °C, and 10 min at 72 °C. For ligation of the common linker, the sample was transferred to ice and 5 µl of PCR linker mixture as in Mueller and Wold(1989), 2 µl of ligation buffer (660 mM Tris-HCl, 50 mM MgCl, 10 mM dithioerythritol, 10 mM ATP, pH 7.5, 20 °C, Boehringer Mannheim), 1 µl of T4 DNA-ligase (5 units/µl, Boehringer Mannheim), and 12 µl of water were added. After an overnight incubation at 4 °C the DNA was ethanol precipitated, washed once with 75% ethanol, dried, and then resuspended in water. The PCR amplification was done in 100 µl of Vent buffer containing 10 pmol each of primer ii and the longer linker primer, 240 µM each dNTP, and 3 units of Vent (exo-) DNA polymerase for 20 cycles using 1 min at 94 °C, 1.5 min at 60 °C, and 3 min at 72 °C. For labeling, the sample was transferred to ice, 5 pmol of T4 kinase [-P]ATP labeled primer iii, 2.5 nmol each dNTP, and 0.5 units of Vent (exo-) DNA polymerase in a volume of Vent buffer not exceeding 15 µl were added. Then the sample was heated to 94 °C for 1.5 min, subjected to 5 cycles of 2 min at 94 °C, 2 min at 62 °C, and 5 min at 72 °C, and kept at 72 °C for 5 more minutes. Samples were transferred to ice, phenol/chloroform extracted, ethanol precipitated, and resuspended in loading dye. One-fifth of each sample was separated on a standard 6% sequencing gel (Sambrook et al., 1989), and the gels were dried and autoradiographed for 15 h at room temperature with Kodak X-Omat LS film.

Visualization of 5`-Ends

Genomic DNA was harvested from unsynchronized Raji cells, and from Raji cells treated with 30 µM aphidicolin for up to 24 h (Saluz and Jost, 1990). 5`-Ends of DNA were visualized by LMPCR on 2 µg of genomic DNA using the same reaction conditions and primer sets as for the in vivo footprints.


RESULTS

Synchronous Growth Arrest of Raji Cells

Initial efforts were focussed on the synchronization of Raji cells. The cells used in our experiments were growing fast, having a doubling time of about 24 h under standard cell culture conditions. For the purpose of arresting cells before and after S phase, we used the drugs mimosine and colchicine, respectively. Colchicine arrests mitotic cells in the metaphase by blocking the spindle apparatus for the separation of the chromosomes (Inoué, 1981). Mimosine, a plant amino acid, has the potential to reversibly block the mammalian cell cycle close to the G/S boundary (Lalande, 1990). The cell cycle block by mimosine may be due to the inhibition of initiation of DNA replication (Mosca et al., 1992). Drugs were added to exponentially growing cells. The flow cytometric analysis of untreated, propidium iodide-stained cells showed a relatively high DNA synthesis rate and a relatively large fraction of the cells in G/M (Fig. 1A). 14 h later, when the cells were harvested for the footprints, a control sample analyzed by flow cytometry showed essentially the same pattern (Fig. 1b). Cells treated with 400 µM mimosine or with 1 µM colchicine for 14 h, respectively, showed a rather different distribution over the cell cycle (Fig. 1, C and D). In the mimosine-treated sample, S phase cells and G/M cells were strongly depleted and a large majority of the cells were in G (Fig. 1C). In the colchicine-treated sample, G cells and S phase cells were strongly depleted and the vast majority of the cells were in G/M (Fig. 1D). These two cell cycle arrests seemed sufficiently quantitative for our intended studies of protein-DNA interactions at DS of oriP before and after S phase.


Figure 1: Histograms of flow cytometric analyses of propidium iodide-stained Raji cells. The relative amount of measured DNA is depicted on the abscissa, the relative amount of cells is shown on the ordinate of each panel. 20,000 events each were gathered and are presented as ungated data. A, untreated, exponentially growing Raji cells at the time of drug addition. The left peak of the histogram represents cells in G, the right peak cells in G/M, cells at various stages of the S phase are in between the two peaks. B-D, 14 h after drug addition: B, untreated control; C, cells treated with 400 µM mimosine; D, cells treated with 1 µM colchicine.



EBNA1 Binding to DS Throughout the Cell Cycle

Cells treated with mimosine and colchicine were harvested and genomic DMS footprints on the initiator element (DS) of oriP of EBV were performed (Fig. 2). The footprinting was done on both strands of DS according to standard methods (Garrity and Wold, 1992; Maxam and Gilbert, 1980; Mueller and Wold, 1989; Saluz and Jost, 1990; Sambrook et al., 1989) with some modifications. The pattern of guanines protected from methylation and nucleotides hypersensitive to methylation is summarized in Fig. 4 . The footprint patterns for mimosine-treated cells and colchicine-treated cells were similar, in general, and consistent with the concept of EBNA1 binding to its four in vitro binding sites (Rawlins et al., 1985) within DS also in vivo (Hsieh et al., 1993). Nuclear proteins other than EBNA1 might also cause the in vivo footprints on the EBNA1 binding sites at some phase of the cell cycle (Oh et al., 1991; Wen et al., 1990). However, since the footprints spanning the EBNA1 binding sites of DS were the same in mimosine- and colchicine-treated cells and also in nontreated, exponentially growing cells (data not shown), we concluded that EBNA1 binding to DS is unchanged throughout the cell cycle (Hsieh et al., 1993). Since EBNA1 binds on only one face of the double helix within DS (Frappier and O'Donnell, 1992; Kimball et al., 1989), the protections of guanines 9037, 9044, 9065, and 9119 are not readily explained solely by the binding of EBNA1. These protections may be caused by structural alterations of the DNA induced by the binding of EBNA1 (Frappier and O'Donnell, 1992), or also by the presence of nuclear proteins other than EBNA1 in the nucleoprotein complexes on DS (Oh et al., 1991; Wen et al., 1990).


Figure 2: Genomic footprinting on both strands of the dyad symmetry element of oriP with dimethyl sulfate. Panel a shows data for the lower strand, panel b for the upper strand. Lanes A+G and G refer to Maxam and Gilbert (1980) sequencing reactions using purified genomic DNA. Lanes Co and Mi are DMS footprints done on live cells treated with colchicine and mimosine, respectively, as shown in Fig. 1. Experiments were done several times from independent synchronous growth arrests with the same results. The numbers on the left of each panel refer to the EBV sequence (Baer et al., 1984), the numbers on the right of each panel refer to the EBNA1 binding sites within DS (Rawlins et al., 1985), nonamers are indicated by the letters A, B, and C.




Figure 4: Summary of genomic footprinting (Fig. 2) and free 5`-ends (Fig. 3) on the dyad symmetry element of oriP of Epstein-Barr virus in Raji cells. The positions of protections and enhanced cleavage sites as presented in Fig. 2 are shown here for G and G/M cells. EBNA1 binding sites (Ambinder et al., 1990) and nonamers (see text) are indicated by horizontal lines drawn between the DNA strands. Numbering of the sequence and the EBNA1 sites refers to Baer et al. (1984) and Rawlins et al. (1985), respectively, the dyad symmetry (Lupton and Levine, 1985; Reisman et al., 1985) is indicated by horizontal arrows, nonamers are labeled A, B, and C. Differences between this genomic sequence and the published B95-8 sequence (Baer et al., 1984) are indicated by small letters above and below the respective DNA strand. Guanines strongly protected from methylation by DMS are indicated by filled circles, weakly protected guanines are indicated by open circles, enhanced reactivity to DMS is shown by filled and open triangles, analogously. The pattern of DMS reactivity for colchicine-treated cells is shown for the entire sequence, the different reactivities of the nonamer in mimosine-treated cells for the upper and lower strand, respectively, are indicated above and below the symbols for reactivities of colchicine-treated cells. Free 5`-ends at nonamers in genomic DNA from untreated cells visualized by LMPCR are indicated by small vertical arrows above and below the respective strand.



Novel Protein Binding at DS

We observed differences between previously published DMS footprints on DS from nonsynchronized cells (Hsieh et al., 1993) and this data. First, there was a guanine instead of an adenine at position 9062 in oriP of our Raji cells, that was methylation protected. This transitional point mutation is unlikely to have any effects on the function of oriP (Ambinder et al., 1990). Second, the adenine at position 9051 was strongly DMS reactive. This difference may be due to variations in technical procedures. The methylation sensitive adenine at 9051 has also been observed in in vitro methylation studies on the distortion of oriP by EBNA1 (Frappier and O'Donnell, 1992). Third, and more important, we observed a novel pattern of DMS reactivity within DS that was not reported by the earlier paper (Hsieh et al., 1993). This discrepancy may be explained by variations in technical procedures or also by differences between Raji cells that have been passaged for many years.

Cell Cycle Dependence

The novel pattern was observed in nonsynchronized cells (data not shown) in a way similar to mimosine-treated cells. However, the pattern was different between mimosine- and colchicine-treated cells. The pattern was found at each nonamer, 5`-TTAGGGTTA-3`, repeated three times within DS. The nonamers are located at 9021 to 9029 (A), 9073 to 9081 (B) and 9127 to 9135 (C), each location being outside of the EBNA1 sites. Nonamer A points to one direction, nonamers B and C point to the other direction. The first guanine of each nonamer was protected stronger in mimosine than in colchicine-treated cells. The third guanine each was slightly stronger methylation sensitive in mimosine- than in colchicine-treated cells. These differences in the footprint patterns between mimosine- and colchicine-treated cells most likely reflect the differential interaction of the nonamer with nuclear protein or, alternatively, a more general change of the nucleoprotein complex on DS. An earlier experimental hint for protein binding to the nonamer sequence came from in vitro footprints on DS. Nuclear proteins from HeLa cells yielded distinct DNase I protected areas within DS that spanned the nonamers (Oh et al., 1991). Therefore, distinct nucleoprotein complexes on DS probably exist in Raji cells in vivo.

DNA 5`-Ends at the Nonamers

In order to correlate more the protein binding data at DS with initiation function, we tried to locate the initiation points of DNA replication within DS at nucleotide resolution. Therefore, we performed LMPCR on purified genomic DNA from untreated Raji cells to visualize DNA 5`-ends. Exponentially growing Raji cells were harvested, genomic DNA was purified, and LMPCR was performed using the same sets of primers as for the genomic footprints. On the lower strand 5`-ends were found mainly at nonamers B and C, on the upper strand mainly at nonamers A and B (Fig. 3, lanes N). The pattern of 5`-ends at the nonamers in untreated DNA is also summarized in Fig. 4. In addition to 5`-ends at the nonamers, 5`-ends were also found upstream of nonamer C for the lower strand (horizontal arrow above C in Fig. 3a) and upstream of nonamer A for the upper strand (two horizontal arrows above A in Fig. 3b) These upstream 5`-ends are less abundant than 5`-ends at the nonamers. They occur at pyrimidine-rich stretches of the respective strands. Since aphidicolin blocks DNA chain elongation (Huberman, 1981), we did the same experiment on genomic DNA from Raji cells treated with aphidicolin. In this experiment the signal for 5`-ends at the nonamers increased strongly in a time dependent fashion (Fig. 3, lanes 24) (only data for 24 h shown). In addition to this strong signal increase at the nonamers, there was also an increased background and an increased abundance of 5`-ends downstream of nonamer C on the upper strand (nicked arrow below nucleotide 9118 in Fig. 3b). These 5`-ends cannot be readily explained by some mechanism, but they may correspond to damaged DNA, resulting from the prolonged treatment of cells with aphidicolin. The increased background of lanes 24 may also result from DNA damage.


Figure 3: Visualization of 5`-ends on both strands of DS in genomic DNA from Raji cells by LMPCR. Panel a shows data for the lower strand, panel b for the upper strand. Lanes A+G and G refer to Maxam and Gilbert (1980) sequencing reactions. Lanes N show DNA 5`-ends occurring in nontreated, nonsynchronously growing cells. Lanes 24 show accumulated 5`-ends in cells treated with 30 µM aphidicolin for 24 h. Experiments were done at least twice with the same results. The numbers on the sides of each panel refer to the EBV sequence, locations of nonamers are indicated by the letters A, B, and C. Horizontal arrows show 5`-ends in genomic DNA from nontreated cells, the nicked arrow indicates an accumulation of 5`-ends in genomic DNA from aphidicolin-treated cells.




DISCUSSION

The EBV latent origin of plasmid replication serves as a model for the controlled replication of eukaryotic chromosomes (DePamphilis, 1988). The only viral protein required for the replication of oriP is EBNA1 (Lupton and Levine, 1985; Yates et al., 1985). Purified EBNA1 lacks detectable ATPase and helicase activity (Frappier and O'Donnell, 1991; Middleton and Sugden, 1992), that both should be needed for the initiation of replication of DNA. Thus, it is likely that at least one cellular protein cooperates with EBNA1 to initiate DNA synthesis at oriP. An important step toward the characterization of cellular proteins required for the initiation of DNA replication from oriP is the examination of in vivo protein-DNA interactions at the initiator element of oriP. A further step is the investigation of in vivo protein binding in dependence on the cell cycle. Therefore, we analyzed in vivo interactions of nuclear proteins with the dyad symmetry element (DS) of oriP of Epstein-Barr virus in Raji cells that were synchronously growth arrested at two points of the cell cycle by the use of mimosine and colchicine. Nucleoprotein complex formation was probed with DMS and visualized by LMPCR. In order to gain a closer view on replication initiation, we tried to map initiation points within DS at nucleotide resolution.

Our results suggest that EBNA1 binds to its 4 binding sites within DS (Lupton and Levine, 1985; Rawlins et al., 1985; Reisman et al., 1985) throughout the cell cycle in an unaltered manner. The same conclusion was made by Hsieh et al.(1993) who performed a study of in vivo protein-DNA interactions on oriP in nonsynchronous Raji cells. In addition, we obtained three novel findings. First, there is in vivo binding activity at three homologous nonamers within the initiator element of oriP of EBV that is apart from EBNA1 binding. Second, the formation of distinct nucleoprotein complexes on DS is possible. The binding at the nonamer may be cell cycle dependent. An analogous situation was recently described for yeast origins of replication. In that case the prereplicative state also showed a more extended footprint than the postreplicative state (Diffley et al., 1994). Third, at the nonamers there are free 5`-ends in untreated genomic DNA which are accumulating under aphidicolin treatment. These 5`-ends may correspond to replication initiation points within DS. A possible replication model would postulate the start points for the leading strand replication for both strands around nonamer B, and the closest lagging strand initiations at and upstream of nonamer A for the upper strand and nonamer C for the lower strand, respectively. In this model the nonamer binding protein would bind before S phase and serve some kind of initiator function. Interestingly, the nonamer sequence 5`-TTAGGGTTA-3` is resembling the human telomeric repeat sequence d(TTAGGG) (Brown, 1989; Cross et al., 1989). Telomeric DNA is able to bind nuclear proteins (Price and Cech, 1987, 1989). Thus, there could be some function for telomere binding proteins or relatives thereof in the replication and nuclear maintenance of oriP containing episomes.

The role of the nonamers has not yet been systematically addressed by functional assays that use plasmids containing scanning mutations in elements A, B, and C of oriP. However, slight deletions into nonamer A disturbed the extrachromosomal maintenance of unrearranged plasmids containing oriP in a plasmid maintenance assay in D98/Raji cells (Chittenden et al., 1989). This functional hint together with our novel data strongly suggest that the nonamer and its binding protein carry out some function in the extrachromosomal maintenance of oriP containing plasmids. A more complete answer, however, on the function of the nonamer elements awaits a thorough mutational analysis of DS, including the nonamers and a biochemical and genetic characterization of the nonamer binding protein.

To our knowledge, this study is the first to use nucleotide resolution genomic footprinting to show a specific, probably cell cycle dependent alteration of the nucleoprotein complex at a viral origin of DNA replication. In addition, the study uses LMPCR to map at nucleotide resolution replication initiation sites from total genomic DNA. This line of research may provide one more step toward the detection of a general mechanism for the replication of chromosomal DNA or for a unique and novel mechanism for the plasmid replication of EBV.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed: Institut für Medizinische Mikrobiologie und Hygiene, Franz-Josef-Strau-Allee 11, D-93053 Regensburg, Germany. Tel.: 49-941-944-6483 or 6413; Fax: 49-941-944-6402; E-mail: Hans-Helmut.Niller@klinik.uni-regensburg.de.

The abbreviations used are: EBV, Epstein-Barr virus; oriP, origin of plasmid replication; S phase, synthesis phase; EBNA1, Epstein-Barr viral nuclear antigen 1; FR, family of repeats; DS, dyad symmetry; LMPCR, ligation mediated polymerase chain reaction; PBS, phosphate-buffered saline; FACScan, fluorescence-activated cell scan; DMS, dimethyl sulfate; dNTP, deoxynucleotide triphosphate; M, mitosis.


ACKNOWLEDGEMENTS

We thank Henryk Lubon for stimulating discussions, and Thomas Dobner, Manfred Marschall, and Udo Reischl for critical reading of the manuscript.


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