Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells
causes distinct changes of SV40 minichromosome-associated
replication proteins
Hans-Jo¨ rg Riedinger, Maria van Betteraey-Nikoleit and Hans Probst
Physiologisch-Chemisches Institut der Universita
¨
tTu
¨
bingen, Germany
Hypoxia interrupts the initiation of simian virus 40 (SV40)
replication in vivo at a stage situated before unwinding of
the origin region. After re-oxygenation, unwinding followed
by a synchronous round of viral replication takes place.
To further characterize the hypoxia-induced inhibition of
unwinding, we analysed the binding of several replication
proteins to the viral minichromosome before and after
re-oxygenation. T antigen, the 34-kDa subunit of replication
protein A (RPA), topoisomerase I, the 48-kDa subunit of
primase, the 125-kDa subunit of polymerase d,andthe
37-kDa subunit of replication factor C (RFC) were present
at the viral chromatin already under hypoxia. The 70-kDa
subunit of RPA, the 180-kDa subunit of polymerase a,and
proliferating cell nuclear antigen (PCNA) were barely
detectable at the SV40 chromatin under hypoxia and signi-
ficantly increased after re-oxygenation. Immunoprecipita-
tion of minichromosomes with T antigen-specific antibody
and subsequent digestion with micrococcus nuclease
revealed that most of the minichromosome-bound T antigen
was associated with the viral origin in hypoxic and in
re-oxygenated cells. T antigen-catalysed unwinding of the
SV40 origin occurred, however, only after re-oxygenation as

The mechanism leading from re-oxygenation to replicon
initiation is largely obscure. The remarkably fast resumption
of initiations after re-oxygenation suggests that the
O
2
-dependent replication control acts very directly on the
replication apparatus.
O
2
-Dependent regulation of replicon initiation was also
demonstrated for viral replication in simian virus 40 (SV40)-
infected CV1 cells [4,5]. As the replication of SV40 is
relatively well investigated, this virus seems to be well suited
to examine the events leading to the reversible shutdown of
replicon initiations by hypoxia. As we have shown, reduc-
tion of the pO
2
to 0.1–0.02% suppresses the viral DNA
synthesis. Re-oxygenation results in new initiations followed
by an almost synchronous round of SV40 replication. This
synchronous round of replication was shown to begin at the
viral origin [4].
After re-oxygenation, the viral replication starts with the
unwinding of the viral origin region [5]. This was shown by
detection of a highly underwound SV40 topoisomer (form
U) about 3 min after re-oxygenation. Form U was not
detectable under hypoxia. Primer RNA-DNA synthesis was
started  3–5 min after re-oxygenation. As form U turned
out to contain primer RNA-DNA, unwinding and primer
synthesis may occur more or less concomitantly after the

replication factor C (RFC), proliferating cell nuclear antigen
(PCNA), and DNA polymerase d [19–21].
In the present communication, we further try to define the
state, at which hypoxia interrupts the initiation of SV40
replication in vivo, by examining which replication proteins
are present in the viral minichromosome before and after
re-oxygenation. The presented data indicate that unwinding
occurs immediately after re-oxygenation but not under
hypoxia. We further demonstrate that a significant fraction
of the proteins engaged in viral replication is bound to the
SV40 minichromosome already under hypoxia, but that
none of the protein complexes necessary for unwinding,
primer synthesis and elongation seems to be completed
before the respective events actually take place.
MATERIALS AND METHODS
Transient hypoxia, re-oxygenation and radioactive
labelling
Monkey CV1 cells (ATCC CCL 70) were grown and
infected with SV40 as described previously [22]. Transient
hypoxia was started 36 h after infection by gassing with
0.04% O
2
/5% CO
2
and Ar to 100% for 6 h [4]. For
re-oxygenation, 0.25 vol. of medium equilibrated with
95% O
2
/5% CO
2

NaHPO
4
,(pH7.0)].The
determination of acid-insoluble radioactivity has been
described previously [23].
Preparation of SV40 minichromosomes
Preparation of SV40 minichromosomes was performed
essentially as described by Su & DePamphilis [24]. In brief,
SV40-infected CV1 cells of Petri dishes 132 mm in diameter
were used for preparation of the minichromosomes.
Stopped cell cultures were washed twice with ice-cold
hypotonic buffer [10 m
M
Hepes/KOH (pH 7.8), 5 m
M
KCl,
0.5 m
M
MgCl
2
,0.1m
M
dithiothreitol]. Cells were homo-
genized with five strokes in a Dounce homogenizer and the
nuclei were pelleted by centrifugation at 3000 g for 5 min.
After resuspension in hypotonic buffer containing protease
inhibitor cocktail (Sigma, Deisenhofen, Germany), the
nuclei were eluted for 1.5 h at 0 °C and then pelleted by
centrifugation at 8000 g for 10 min. The minichromosomes
in the supernatant were sedimented at 14 000 g in a

NaOH, 0.6
M
NaCl, 1 m
M
EDTA, 0.1% sodium lauroylsarcosinate. After centrifu-
gation in a Beckman SW40 rotor at 164 000 g and 23 °C
for 16 h, 0.6-mL fractions were collected from the top of
the gradient and analysed for acid-insoluble radioactivity
[23].
Electrophoresis of minichromosome-bound proteins,
Western blotting
Minichromosomes resuspended in NaCl/P
i
were diluted
with 10 vol. of 10 m
M
sodium pyrophosphate and 10 m
M
EDTA (pH 8.0), and proteins were extracted with 4 vol. of
phenol (pH 8.0). The phenolic phase was then extracted
twice with the same volume of 10 m
M
sodium pyrophos-
phate, 10 m
M
EDTA (pH 8.0) and proteins were precipi-
tated by addition of 5 vol. of acetone at )20 °Covernight.
After centrifugation at 200 000 g,4°C for 45 min, the
pellet was successively washed with chloroform/CHCl
3

r medizinische Biochemie, Jena,
Germany), 1 : 1000; RFC (polyclonal antibody against
the 37-kDa subunit, a kind gift of J. Hurwitz, Memorial
2384 H J. Riedinger et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Sloan Kettering Cancer Center, New York, USA), 1 : 1000;
PCNA (monoclonal antibody, clone 19F4, Roche,
Mannheim, Germany) 2 lgÆmL
)1
; polymerase d (monoclo-
nal antibody against the 125-kDa subunit, Transduction
laboratories, Heidelberg, Germany) 1 : 1000.
Digestion of immunoprecipitated minichromosomes
with micrococcus nuclease and correlation
of the digestion products with the SV40 genome
SV40-infected cells were incubated hypoxically for 6 h and
simultaneously labelled with [methyl-
3
H]deoxythymidine
(10 lCiÆmL
)1
). Thereafter, cells were either stopped or
re-oxygenated for 6 min and then stopped. Minichromo-
somes were isolated as described above. For immunopre-
cipitation, 10 mL of T antigen-specific hybridoma
supernatant (clone pAB 101) were incubated with pro-
tein A agarose (20 mg, Biorad, Richmond, USA) for 1 h
at 4 °C. Thereafter, protein A agarose was pelleted and
minichromosomes were bound by incubation for 90 min at
4 °C in NET buffer [150 m
M

(1.5
M
NaCl, 0.15
M
sodium citrate, pH 7.0), heated to
65 °C for 10 min and chilled on ice. The DNA was then
transferred to the membrane (2 cm in diameter), dried at
37 °C and fixed by UV irradiation. Hybridization was
performed at 37 °C, as described previously [27]. After
hybridization, the radioactivity bound to each of 14
probes, representing both complementary DNA strands
of seven SV40 genome fragments (see inset in Fig. 3), was
quantified and normalized for size of the respective SV40
segment.
Nuclease P1 digestion of SV40 minichromosomes
SV40 minichromosomes isolated from hypoxic and
re-oxygenated cell cultures were digested with nuclease P1
essentially as described by Adachi & Laemmli [28]. After
sedimentation, the minichromosomes were redissolved in
HMN buffer [5 m
M
Hepes/NaOH (pH 7.5), 8 m
M
MgCl
2
,
100 m
M
NaCl] and one half was digested with 1 U of
nuclease P1 [Pharmacia, Freiburg, Germany; 1 UÆlL

3
and dialyzed
against 1 m
M
Tris/0.1 m
M
EDTA (pH 8.0) at 4 °Cover-
night. After digestion with RNase A (100 lgÆmL
)1
at 37 °C
for 1 h), 100 ng of isolated DNA per slot was loaded onto a
25 · 20 cm agarose gel containing 20 lgÆmL
)1
chloroquine
in gel buffer (30 m
M
NaH
2
PO
4
,36m
M
Tris, 1 m
M
EDTA).
Electrophoresis was carried out at 2 VÆcm
)1
and 4 °Cfor
20 h. Southern blotting was performed under alkaline
conditions [26]. The DNA was detected by hybridization

spheric pO
2
or re-oxygenated after 6 h hypoxia for 10 or
25 min and labelled with [methyl-
3
H]deoxythymidine dur-
ing the last 10 min of the incubation. SV40 minichromo-
somes and resuspended eluted nuclei were brought to 0.2
M
NaOH and incubated for 1 h at room temperature.
3
H-Labeled DNA in the lysates was then analysed for size
by alkaline sucrose gradient centrifugation. As a control,
noninfected, normoxically cultivated CV1 cells where trea-
ted exactly in the same way.
Figure 1 shows that there were no significant differences
between the peak positions, i.e. the lengths of growing DNA
strands, in minichromosomes and nuclei. Incubating cell
cultures under atmospheric pO
2
(Fig. 1A,B) resulted in
peaks around fraction 10, representing full-length SV40
DNA. Additionally, a peak at fraction 18, representing
Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur. J. Biochem. 269) 2385
covalently closed, supercoiled SV40 DNA was visible.
Accordingly, this peak was predominant in long-term
labelled DNA (circles). Minichromosomes containing
long-term labelled (supercoiled) SV40 DNA seemed to
preferably remain in the nuclei, as only 10–20% were eluted
compared to 30–50% of the pulse-labelled (replicating)

prepared, separated by SDS/PAGE and blotted to a
nitrocellulose membrane. Chosen proteins were then detec-
ted with specific antibodies. As quantification of Western
blots is known to be difficult, reproducibility of the results
was carefully ascertained, especially in cases where changes
before and after re-oxygenation were found to be small (e.g.
for T antigen or polymerase a-180). Each protein was tested
in at least three independent experiments and representative
results are shown in Fig. 2.
RPA-34, topoisomerase I, primase-48, RFC-37, and
polymerase d-125 were associated with the SV40 minichro-
mosomes already under hypoxia. For RPA-34 and RFC-37,
longer exposure times were chosen to accentuate slower
migrating bands. If at all, only minor differences in signal
intensities were found for these proteins irrespective whether
they were isolated from minichromosomes of hypoxic or
re-oxygenated cell cultures. In case of RPA-34 and RFC-37,
this was confirmed by shorter exposure (data not shown).
Topoisomerase I, the 34-kDa subunit of RPA and the
37-kDa subunit of RFC seemed to exist in several modi-
fications (Fig. 2). Two dimensional gel electrophoresis (first
dimension: isoelectric focusing; second dimension: SDS/
PAGE) of SV40 minichromosome-bound proteins, Western
blotting and immunodetection revealed that, for RPA-34
and RFC-37, these modifications differed significantly in
their isoelectric points but only slightly in their molecular
masses (data not shown). This suggests that they are
differently phosphorylated species. The patterns of topo-
isomeraseandRFC-37werethesameirrespectiveofthe
Fig. 1. Alkaline sucrose gradient centrifugation of viral DNA of mini-

different phosphorylation forms of the protein. Rather, the
lower band may be a degradation product of the upper.
Recently, Schumacher et al.[29]isolatedaN-terminal
truncated form of polymerase d with an apparent molecular
mass of 116 kDa, approximately the same size as the lower
band of polymerase d detected by us.
The ratio of the intensities of both bands significantly
changed. The upper band increased 3 and 5 min after
re-oxygenation, while the lower band decreased. The sum of
the bands remained, however, largely constant. This was
especially evident in preparations that yielded essentially
equal signal intensities of both bands from the beginning.
Supposing the lower band to be a degradation product of
the upper, this result could indicate that polymerase d was
better protected against degradation when it was stably
bound to its target at the replication fork, i.e. during primer
elongation.
An unexpected binding behaviour showed T antigen.
Under hypoxia, it was associated with the chromatin but
decreased immediately after re-oxygenation to about one
third. Three minutes after re-oxygenation, the amount of
minichromosome-bound T antigen increased again and
attained the level seen under hypoxia until 5 min after
re-oxygenation.
The 70-kDa subunit of RPA, the 180-kDa subunit of
DNA polymerase a, and PCNA were barely detectable in
hypoxic minichromosomes. After re-oxygenation, the
amount of all three proteins significantly increased, though
at different times. RPA-70 increased after just 1 min and
further increased up to 3 min, remaining then at a

showninFig.3.
DNA isolated from the immunoprecipitate, i.e. DNA
fragments that were associated with T antigen before
purification, mainly hybridized to SV40 probes 1a and 1,
irrespective whether cells were re-oxygenated for 6 min or
kept hypoxic (Fig. 3A,C). This means that T antigen is
preferably bound to the core origin (represented by
probe 1a) or to the whole SV40 origin region (represented
by probe 1). After re-oxygenation, but not under hypoxia,
some DNA also hybridized to probe 4, indicating that
T antigen was also associated with SV40 DNA containing
the termination region at this time. As 6 min of
re-oxygenation are not sufficient to allow complete replica-
tion of the whole SV40 genome [4,5], association of T
antigen with this fragment likely results from elongation of
SV40 replicons, which were started, but not yet terminated,
before or during hypoxia.
DNA fragments isolated from the supernatant of the
immunoprecipitation hybridized more or less uniformly to
all probes except 1a (Fig. 3B,D). As 1a represents the SV40
core origin, this result indicates that, in most minichromo-
somes, the core origin is protected against digestion with
micrococcus nuclease by association with T antigen.
Fig. 2. Immunodetection of minichromosome-
associated replication proteins. Minichromo-
some-bound proteins isolated from hypoxic
(H) or re-oxygenated (re-oxygenation times
are indicated) SV40-infected cell cultures were
separated by SDS-PAA gel electrophoresis,
Western blotted and detected with appropriate

hypoxia. Smaller local distortions, which were detected after
binding of T antigen to SV40 site II at the early palindrome
and the AT-rich region in vitro [6–11], may exist under
hypoxia. They may, however, be protected against digestion
by micrococcus nuclease through the origin-bound T
antigen hexamer. In vitro DNase protection assays demon-
strated that the entire 64-bp core origin is protected against
digestion by DNase I, when T antigen is bound [6,35].
After re-oxygenation, the minichromosomes became
more sensitive to nuclease P1 digestion, resulting in an
increase of nicked open circle DNA and in a decrease of
supercoiled DNA compared to the undigested fraction. P1
susceptibility of the minichromosomes became apparent
just 1 min after re-oxygenation and remained constant for
longer re-oxygenation times, indicating that the length of
the unwound DNA stretch is not decisive for P1 digestion in
Fig. 3. Localization of T antigen at the SV40
chromatin.
3
H[thymidine]-labelled minichro-
mosomes isolated from hypoxic (A,B) or
re-oxygenated (C,D) SV40-infected CV1 cells
were immunoprecipitated with T antigen
antibody-saturated protein A agarose. The
immunoprecipitate was resuspended and
digested with micrococcus nuclease. After
centrifugation, DNA fragments were isolated
from the immunoprecipitate and the super-
natant and analysed by hybridization against
membrane-fixed single-stranded M13mp18

chromosomes, despite of the facts that RPA-70 was barely
detectable (Fig. 2) and that the viral genome was obviously
not unwound before re-oxygenation (Fig. 4). As the inter-
action between RPA and single-stranded DNA is largely
mediated by RPA-70 [36,37], these observations together
indicate that RPA-34 is bound to the viral chromosome by
protein–protein interactions or by any other means than
interaction with single-stranded DNA under hypoxic
culture conditions. Following re-oxygenation, part or all
of the prebound RPA-34 may become integrated into the
RPA heterotrimer complex.
In order to examine which amount of RPA-34 was bound
to unwound SV40 minichromosome DNA in form of a
RPA heterotrimer complex before and after re-oxygenation,
we made use of the fact that, once bound to single-stranded
DNA, the resulting complex cannot be disrupted by excess
single-stranded competitor DNA [28]. RPA-34 bound by
other means to the SV40 minichromosome, on the other
hand, is readily displaced by excess competitor DNA
(see below), probably because it contains a single-stranded
DNA-binding motif [38,39].
We therefore treated minichromosomes isolated from
hypoxic or re-oxygenated cell cultures with an excess of
single-stranded herring sperm DNA (Fig. 5B) and com-
pared the dissociation of RPA-34 with the respective
untreated control (Fig. 5A). RPA-34 was found to be
almost totally displaced from minichromosomes of hypoxic
cell cultures. Minichromosomes from cells re-oxygenated
for longer than 3 min, on the other hand, retained part of
their RPA-34. Higher phosphorylated forms of RPA-34

replication after re-oxygenation, we tested the influence of
these inhibitors on the formation of SV40 form U, which
Fig. 4. Sensitivity of minichromosomes of hypoxic and re-oxygenated
cell cultures to single-stranded DNA-specific nuclease P1. Aliquots of
SV40 minichromosomes of hypoxic or re-oxygenated cells were
immunoprecipitated with immobilized T antigen-specific antibody
with or without prior digestion with P1 nuclease (1 U). SV40 DNA
was isolated, separated on an agarose gel, blotted and detected by a
32
P-labeled DNA-probe. Signal intensities of open circle and super-
coiled SV40 DNA-bands were quantified by densitometric evaluation.
The signal intensities of the undigested aliquots were set as 100%. d,
signal intensity of open circle DNA (oc); j,signalintensityofsuper-
coiled DNA (sc). H, hypoxic.
Fig. 5. Dissociation of minichromosome-bound RPA-34 by excess
competitor single-stranded DNA. Minichromosomes of hypoxic or
re-oxygenated SV40-infected cell cultures were incubated without (A)
or with (B) 40 lgÆmL
)1
of denatured herring sperm DNA for 30 min
at 30 °C. Minichromosome-bound proteins were isolated, separated
by SDS/PAGE and Western blotted. The RPA subunits were detected
with specific antibodies. Exposure times were the same for (A) and (B).
Re-oxygenation times are indicated. H, hypoxic cells.
Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur. J. Biochem. 269) 2389
was shown to be a product of the unwinding of the viral
origin region in vivo [5]. The inhibitors were added to the
hypoxically cultivated cell cultures 30 min before re-oxy-
genation. After re-oxygenation for 6 min, whole-cell DNA
was isolated and separated on a chloroquine containing gel.

were bound to the viral minichromosome already before
re-oxygenation. RPA-70, polymerase a-180, and PCNA
were barely detectable under hypoxia and increased signi-
ficantly after re-oxygenation (Fig. 2). The small amounts of
these proteins, which were detectable under hypoxia, may
be bound to originally replicative viral chromosomes, which
were no more replicated during the hypoxic period [4].
These chromosomes amount up to 5% of the replication-
competent viral chromatin.
In vitro studies on SV40 DNA replication suggest that the
following steps occur during initiation. First, in an ATP-
dependent reaction, SV40 large T antigen binds as a double
hexamer to the viral origin, leading to local distortions in the
AT-rich region and partial melting of the early palindrome
[7–11]. The denatured DNA in the early palindrome is then
bound by RPA, possibly at the pyrimidine-rich top strand
[8,13,48,49]. RPA is first associated in form of an unstable
complex (RPA
8nt
), in which it binds eight nucleotides of
single-stranded DNA [13,30,31]. After further unwinding of
the origin, including part of the central palindrome, RPA
8nt
turns into a stable complex (RPA
30nt
), in which RPA
contacts about 30 nucleotides of single-stranded DNA.
Formation of the RPA
30nt
-complex may be followed by

1, hypoxic cells, not re-oxygenated; 2, cells re-oxygenated without
inhibitor; 3, cells re-oxygenated in the presence of staurosporine; 4,
cells re-oxygenated in the presence of olomoucine; 5, cells re-oxygen-
ated in the presence of wortmannin. LC, late Cairns SV40 DNA; IC,
intermediate Cairns SV40 DNA; T, topoisomers of mature SV40
DNA (form I); U, form U.
2390 H J. Riedinger et al. (Eur. J. Biochem. 269) Ó FEBS 2002
result, KMnO
4
footprinting experiments of the SV40 core
origin region revealed no differences between hypoxic and
re-oxygenated minichromosomes (data not shown). What
may be the reason that T antigen remains bound to the SV40
origin after initiation of replication? Several explanations are
possible. First, initiation of replication upon re-oxygenation
is not exactly synchronous. Secondly, only a part of the T
antigen-containing minichromosomes actually replicate,
and thirdly, the duplicated origin may be rebound by free
T antigen, leading to restoration of the state before
re-oxygenation. The last mentioned notion is supported by
the observation that, after a significant decrease 1 min after
re-oxygenation, minichromosome-bound T antigen was
found to be re-elevated 3 and 5 min after re-oxygenation
(Fig. 2).
Unwinding of the viral origin, primer synthesis and primer
elongation are dependent on re-oxygenation. This has been
shown previously [5] and is also demonstrated by the results
presented here. The data indicate that unwinding is initiated
as soon as 1 min after re-oxygenation. (a) The minichromo-
somes were sensitive to single-stranded DNA-specific nuc-

hypoxia (RPA-34, topoisomerase, primase-48, RFC-37,
polymerase d-125). Remarkably, individual subunits of
protein complexes, generally believed to act only in a
complexed form, e.g. RPA-34 and -70, primase and
polymerase a, or RFC, PCNA and polymerase d, behave
differently in this respect. Murti et al. [50] found that the
RPA subunits also partition differently during the cell cycle.
The authors demonstrated that the subunits colocalized
only during the G1- and S-phase of the cell cycle. During
mitosis, the subunits dissociated and partitioned into
different cell compartments; p34 was found at the
chromosomes, p70 at the spindle poles and p11 in the
cytoplasm. Despite of the fact that hypoxic SV40-infected
cells probably never leave a S-phase-like state, these results
show that the different subunits of RPA are not necessarily
assembled in the heterotrimer complex but may also exist as
single proteins in the living cell.
Altogether, the presented results situate the hypoxia-
induced block of SV40 replication somewhere between
binding of the T antigen to site II and unwinding of the viral
origin. The mechanism, which triggers the release of the
block following re-oxygenation, remains unclear. Clearly,
the period between re-oxygenation and beginning of
unwinding is too short to allow changes in gene expression
or other time-consuming processes. The possibility that
some proteins have to be synthesized before initiation is
ruled out by the fact that inhibition of protein biosynthesis
by emetine immediately before re-oxygenation has no
influence on unwinding and the following viral replication
round (data not shown). Moreover, all replication proteins

123 were phosphorylated or not. Effective unwinding is also
influenced by phosphorylation of Thr124 of T antigen,
probably catalysed by cyclin A/cdk2 [53–56]. As neither
staurosporine nor olomoucine, both inhibitors of cyclin A/
cdk2, inhibit formation of form U, i.e. unwinding, after
re-oxygenation (Fig. 6A), it seems likely that Thr124 is
phosphorylated before re-oxygenation.
Alternatively, binding of transcription factors like AP1 or
NFjB may be necessary to activate unwinding after
re-oxygenation [57–61].
In a recent study, we have shown that glucose in
millimolar concentrations prevents hypoxia-induced inhibi-
tion of SV40 DNA replication in infected CV1 cells [62]. As
we have outlined in this study, though O
2
and glucose are
main substrates for cellular ATP generation, it seems
Ó FEBS 2002 Initiation of SV40 replication in vivo (Eur. J. Biochem. 269) 2391
unlikely that ATP shortage is responsible for inhibition of
SV40 replication under hypoxia. This was suggested by the
finding that the ATP concentration remained unchanged in
SV40-infected CV1 cells during a 4-h hypoxic incubation,
whereas unwinding, as determined by generation of SV40
form U, and incorporation of [methyl-
3
H]deoxythymidine
into DNA declined to below 20% of control levels.
Nevertheless, we cannot exclude the possibility that while
overall cellular ATP concentration remains constant, chan-
ges of ATP concentration in cellular compartments, e.g. the

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