From the
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
Plasmid replication of Epstein-Barr virus (EBV)
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)
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.
We thank Henryk Lubon for stimulating discussions, and
Thomas Dobner, Manfred Marschall, and Udo Reischl for critical reading
of the manuscript.
-Allee 11, D-93053 Regensburg, Germany
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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.
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.
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.
(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.
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.