Báo cáo khoa học: Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase ppt

Initiation of JC virus DNA replication
in vitro
by human and mouse
DNA polymerase a-primase
Richard W. P. Smith
1,
* and Heinz-Peter Nasheuer
1,2
1
Abteilung Biochemie, Institut fu
¨
r Molekulare Biotechnologie, Jena, Germany;
2
National University of Ireland, Galway,
Department of Biochemistry, Galway, Ireland
Host species specificity of the polyomaviruses simian virus
40 (SV40) and mouse polyomavirus (PyV) has been shown
to be determined by the host DNA polymerase a-primase
complex involved in the initiation of both viral and host
DNA replication. Here we demonstrate that DNA repli-
cation of the related human pathogenic polyomavirus JC
virus (JCV) can be supported in vitro by DNA polymerase
a-primase of either human or murine origin indicating that
the mechanism of its strict species specificity differs from
that of SV40 and PyV. Our results indicate that this may
be due to differences in the interaction of JCV and SV40
large T antigens with the DNA replication initiation
complex.
Keywords: DNA replication; initiation; DNA polymerase
a-primase; species specificity; polyomavirus.
Polyomavirus DNA replication has served as a model

system to study eukaryotic DNA replication [1,2]. JC virus
(JCV) belongs to the polyomavirus family and is the
causative agent of progressive multifocal leukoencephalo-
pathy in immunocompromised humans (reviewed in [3–7]).
JCV exhibits a highly restricted host range and this species
specificity appears to be governed by host encoded DNA
replication factors as hamster glial cells, which support viral
early gene transcription, nevertheless fail to replicate JCV
DNA [8]. JCV is closely related to simian virus 40 (SV40)
and to mouse polyomavirus (PyV), both of which show
clear species specificities as lytic infection is limited to
primate and mouse cells, respectively [9]. The species
specificities of both SV40 and PyV are regulated at the
level of initiation of DNA replication [10], a process that
has been extensively studied both in vivo and in vitro owing
to the development of cell-free DNA replication systems
[2,11–16].
Polyomavirus DNA replication is carried out by the host
cell machinery supplemented with a single essential viral
protein, large T antigen (TAg), which recognizes and
partially unwinds the viral replication origin, recruits host
proteins such as replication protein A (RPA) and DNA
polymerase a-primase, and functions as the replicative
helicase [2,17,18]. Species specificity of both SV40 and PyV
DNA replication can be reproduced in vitro using DNA
carrying the viral core origin and purified replication
enzymes [14,19–21]. For both SV40 and PyV it has been
clearly demonstrated that the host factor responsible for
species specificity is DNA polymerase a-primase, which
initiates DNA replication in all eukaryotes [19,22–27].

DNA polymerase a-primase consists of four subunits with
apparent molecular masses of 180, 68, 58 and 48 kDa of
which the largest and smallest subunits are a DNA
polymerase and a primase, respectively [28–30]. SV40
DNA replication in vitro was recently shown to require a
functional interaction between the SV40 TAg and the
C-terminus of the p180 subunit of human DNA poly-
merase a-primase [26].
The genome of JCV is 69% homologous to that of SV40
and expresses an analogous set of proteins [31]. The core
origins of replication of the two viruses are also conserved to
such an extent that SV40 TAg, which is 72% identical to
JCV TAg, can efficiently support JCV DNA replication
in vivo and in vitro [32,33]. The high level of conservation
between these two primate specific viruses coupled with the
fact that JCV DNA replication is inhibited in a nonpermis-
sive host in vivo would imply that the restricted host range of
JCV is due to a requirement for human DNA polymerase
a-primase, as is the case with SV40 [8]. Previously we
reported the establishment of a cell-free system for JCV
DNA replication [32]. With this system we were able to
reproduce many features of JCV DNA replication found
in vivo, such as sequence requirements at the origin of
replication and the requirement for JCV or SV40 (but not
PyV) TAg for efficient replication. Therefore, we applied
this system to the question of host specificity regulation and
found that this differs from that of SV40 in that it is not
determined at the level of initiation of DNA replication by
DNA polymerase a-primase in vitro. This appears to be due
to differences in the interaction of the JCV and SV40 large

T antigens with the initiation complex.
Correspondence to H. P. Nasheuer, National University of Ireland,
Galway, Department of Biochemistry, Cell Cycle Control
Laboratory, Galway, Ireland.
Fax: + 353 91 512 504, Tel.: + 353 91 512 409,
E-mail:
Abbreviations: JCV, JC virus; SV40, simian virus 40; PyV, mouse
polyomavirus; TAg, large T antigen; RPA, replication protein A.
*Present address: Institute of Virology, University of Glasgow,
Church Street, Glasgow G11 5JR, Scotland UK.
(Received 30 October 2002, revised 4 March 2003,
accepted 17 March 2003)
Eur. J. Biochem. 270, 2030–2037 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03579.x
Materials and methods
Proteins
SV40 TAg, JCV TAg and the DNA polymerase
a-primase complexes (p180-p68-p58-p48) were purified
from baculovirus infected insect cells as described previously
[22,32,34,35]. Human and murine DNA polymerase
a-primase were immunopurified using the monoclonal
antibodies SJK237-71 and SJK287-38, respectively [36].
Human RPA was bacterially expressed and purified as
outlined previously [37,38]. Human topoisomerase I was
expressed in insect cells and purified as described by Søe
et al. [39] and was a generous gift of K. Søe (IMB-Jena,
Germany). The monoclonal antibodies SJK237-71 and
SJK287-38, specific for DNA polymerase a-primase, were
purified by affinity chromatography [40]. Protein concen-
tration was determined according to Bradford [41] using a
commercial reagent with BSA as a standard (Biorad,

Munich). DNA polymerase a and DNA primase assays
were performed as previously described [34,42,43].
Preparation of S100 extracts and replication
of SV40 and JCV
in vitro
S100 extracts were prepared from logarithmically growing
FM3A cells as previously described [27,34]. Cells were
harvested by centrifugation, then washed twice with phos-
phate buffered saline (NaCl/P
i
) and once with hypotonic
buffer. The cells were resuspended in hypotonic buffer,
incubatedfor10minonice,andbrokenby12strokesina
Dounce homogenizer. The extracts were centrifuged at 4 °C
and 11 000 g. The supernatant was then adjusted to
100 m
M
NaCl and clarified by a second centrifugation at
100 000 g (S100 extract). Depletion of DNA polymerase
a-primase from S100 extracts was performed essentially as
previously described [22,27,32].
The replication of SV40 and JCV DNA in vitro was
performed as previously described [22,27,32]. Briefly, the
assay contained 0.6 lg SV40 or JCV TAg, 250 ng of pUC-
HS or pJC389 or pJC433 DNA (carrying the replication
origin of SV40 or JCV, respectively [21,32]), and 200 lg
S100 in 30 m
M
Hepes/NaOH (pH 7.8), 1 m
M

dithiothreitol,
7m
M
magnesium acetate, 1 m
M
EGTA (pH 7.8), 4 m
M
ATP, 0.3 m
M
CTP, GTP, and UTP, 0.1 m
M
dATP and
dGTP, 0.05 m
M
dCTP and dTTP, 40 m
M
creatine phos-
phate, and 80 lgÆmL
)1
creatine kinase, and 5 lCi each of
[a
32
P]dCTP and [a
32
P]dTTP (3000 CiÆmmol
)1
, Amersham-
Biosciences). DNA polymerase a-primase was added as
indicated. The incorporation of radioactive dNMP was
measured by acid-precipitation of DNA and scintillation

counting. The total radioactivity was measured after
spotting 5 lL of a 200-fold dilution of the replication assay
onto GF52 filters (Schleicher & Schu
¨
ll, Dassel, Germany).
EcoRI and DpnI digestion of product DNA was carried out
as described by Kautz et al. [23].
Initiation of replication on JCV DNA
Initiation reactions were performed essentially as previously
described [22,32,44,45]. Briefly, the JCV initiation assay
(40 lL) was assembled on ice and contained 0.25 lg
pJC389 (carrying the JCV replication origin), 0.6 lgJCV
TAg, and 0.5 lgRPA,in30m
M
Hepes/KOH (pH 7.8),
7m
M
magnesium acetate, 1 m
M
EGTA, 1 m
M
dithiothre-
itol, 0.2 m
M
UTP, 0.2 m
M
GTP, 0.01 m
M
CTP, 4 m
M

ATP,
40 m
M
creatine phosphate, 1 lg creatine kinase, 0.3 lg
topoisomerase I, 0.25 mgÆmL
)1
heat treated BSA, and
20 lCi of [a-
32
P]CTP (3000 CiÆmmol
)1
, NEN Life Science,
Brussels). Recombinant DNA polymerase a-primase was
added as indicated in the figure legends. After incubation for
2 h at 37 °C one-eighth of the reaction mixture was spotted
onto DE81 paper to estimate the amount of incorporated
nucleotides [46]. For size analysis the reaction products were
precipitated with 0.8
M
LiCl, 10 lg of sonicated salmon
sperm DNA (Sigma), 10 m
M
MgCl
2
and 120 lL of ethanol
for 1 h on dry ice, washed twice with 75% ethanol/water,
dried, redissolved in 45% formamide/5 m
M
EDTA/0.05%
(w/v) xylene cyanol FF/0.05% (w/v) bromphenol blue at

65 °C for 30 min, heated for 3 min at 95 °C, and electro-
phoresed in denaturing 20% polyacrylamide gels for 3–4 h
at 600 V as described previously [22]. The reaction products
were visualized by autoradiography and quantified with a
phosphoimager (Amersham Biosciences).
JCV and SV40 monopolymerase systems
These assays were adapted from Ishimi et al.[11].The
monopolymerase assay (40 lL) was assembled on ice and
contained 0.5 lg pUC-HS (carrying the SV40 replication
origin) or 0.5 lg pJC433 (carrying the JCV replication
origin [32]), 1 lg SV40 or JCV T antigen, 1.4 ng topoiso-
merase I and 0.5 lgRPA,in30m
M
Hepes/KOH (pH 7.8),
7m
M
magnesium acetate, 0.1 m
M
EGTA, 1 m
M
dithio-
threitol, 0.2 m
M
UTP, 0.2 m
M
GTP, 0.2 m
M
CTP, 4 m
M
ATP, 20 m

M
dATP, 20 m
M
dGTP, 2 m
M
dCTP, 2 m
M
dTTP, 40 m
M
creatine phosphate, 1 lg creatine kinase,
0.25 mgÆmL
)1
heat treated BSA, and 5 lCi [a-
32
P]dCTP
and 5 lCi [a-
32
P]dTTP (each 3000 CiÆmmol
)1
, Amersham-
Biosciences). Recombinant DNA polymerase a-primase
was added as indicated. After incubation for 90 min at
37 °C, one-quarter of the reaction mixture was used to
determine the level of incorporation by spotting onto DE81
paper, washing with 0.5
M
NaHCO
3
and liquid scintillation
counting [46].

Results
Replication of JCV DNA in crude cell extracts with
recombinant human and murine DNA polymerase
a-primase
Previously we reported the establishment of in vitro systems
for the replication of JCV DNA, either using crude cell
extracts or purified proteins only [32]. Both these systems
were dependent upon recombinant JCV TAg and the
presence of a JCV origin of DNA replication. Here we
applied these systems to study the dependence of JCV DNA
replication on human replication proteins. Figure 1 repre-
sents a comparison between the SV40 (panel A) and JCV
(panel B) cell-free DNA replication systems. Figure 1B
shows a DNA replication assay using mouse FM3A S100
crude cell extracts depleted of DNA polymerase a-primase
supplemented with JCV TAg, a plasmid carrying the JCV
Ó FEBS 2003 Initiation of DNA replication (Eur. J. Biochem. 270) 2031
replication origin (pJC389) and either recombinant human
or murine DNA polymerase a-primase expressed using the
baculovirus system (Fig. 1B, columns 1–7). The replication
activity of JCV TAg in mouse cell extracts is not dependent
on the sequence of the plasmid as the incorporation of
radioactive dNMPs was the same whether the plasmid
pJC389 or pJC433 was used in the cell-free replication assay
(data not shown).
In parallel, we show an SV40 DNA replication assay
using the same cell extracts but with SV40 TAg and a
plasmid (pUC-HS) that carries the SV40 replication origin
(Fig. 1A, columns 1–7 [26,27]). As we showed previously,
SV40 DNA replication is absolutely dependent upon human

DNA polymerase a-primase (Fig. 1A, columns 1–5 [26,27])
and more specifically, upon the human p180 subunit as the
hybrid DNA polymerase a-primase complex consisting of
the murine p180 subunit together with the human p68, p58
and p48 subunits (MH
3
) is inactive (Fig. 1A, columns 6–7
[26,27]). In contrast, replication of JCV DNA does not show
the same strict requirement for human DNA polymerase
a-primase (Fig. 1B, columns 1–7). Both murine DNA
polymerase a-primase and the hybrid complex, MH
3
, show
significant activity in the DNA replication assay. In both the
SV40 and JCV assays, incorporation of nucleotides is
dependent upon an intact origin of replication; either
plasmids carrying the noncognate PyV origin or a disabled
JCV origin are inactive (Fig. 1A, column 8 and Fig. 1B,
columns 8 and 9). Background values determined without
added DNA polymerase a-primase were higher in the JCV
system, presumably due to residual endogenous murine
enzyme in the cell extracts due to incomplete depletion.
However, as this background incorporation did not repre-
sent full rounds of replication in either system (Fig. 2A,B,
columns 1–2) other relevant values (Fig. 1A,B, lanes 2–7)
were corrected for it.
We further characterized the products of the replication
reactions by digesting the resulting DNA with the restriction
enzyme DpnI, which digests only fully methylated DNA.
The plasmid DNA used as template in our assay was

purified from Escherichia coli and is therefore fully methy-
lated. However, one or more full rounds of replication will
result in hemimethylated or unmethylated products and will
consequently lead to DpnI resistance which is indeed
observed after replication of JCV DNA either with human
or with murine DNA polymerase a-primase (Fig. 2B, lanes
4 and 6). The lack of species specificity we observed was
reproducible with various independently expressed and
purified batches of JCV TAg (data not shown) and with
various batches of the template DNAs pJC389 and pJC433
([32]; Figs 1 and 2). As expected murine DNA polymerase
a-primase did not support SV40 DNA replication (Fig. 2A,
lanes 5 and 6).
JCV DNA replication with purified proteins
We note that in the cell-free system JCV DNA replication is
markedly less efficient when driven by murine compared
with human DNA polymerase a-primase. In order to
Fig. 1. DNA replication assays in murine FM3A cell extracts depleted of DNA polymerase a-primase using the SV40 (A) and JCV (B) systems.
(A) The SV40 system makes use of SV40 TAg and pUC-HS template DNA containing the SV40 origin of DNA replication. (B) The JCV system
uses JCV TAg and pJC389 DNA with the JCV origin. The cell extracts were supplemented with 0.5 and 1.0 DNA polymerase units of the indicated
DNA polymerase a-primase complexes (H
4
, human heterotetramer; M
4
, murine heterotetramer; MH
3
, murine p180 with human p68, p58 and p48).
Enzyme activities were determined beforehand with a DNA polymerase assay on activated calf thymus DNA. (A) and (B) column 1, TAg omitted;
column 8, pJC389I-/II-, containing a disabled JCV replication origin [32], was used as template for human DNA polymerase a-primase with SV40
(A) and JCV TAg (B), respectively. (B) column 9, pUC-Py1, containing the PyV replication origin [21], used with human DNA polymerase

a-primase and JCV TAg. Values in panels (A) and (B), columns 2–7 were corrected for background incorporation determined in either system
without the addition of DNA polymerase a-primase. Standard deviations from two to four experiments are indicated as error bars. The experiments
in (A) and (B) were performed in parallel.
2032 R. W. P. Smith and H. P. Nasheuer (Eur. J. Biochem. 270) Ó FEBS 2003
characterize further these processes we made use of purified
systems. Firstly we examined the efficiency of primer
formation at the origin of replication during the initiation
step of JCV DNA replication. Figure 3 shows that this is
efficiently carried out by both the human and murine DNA
polymerase a-primase complexes although quantification of
the reaction products shows the murine complex to be
approximately 30% less efficient than the human complex,
especially in the synthesis of products greater than four
nucleotides in length. Murine DNA polymerase a-primase
is absolutely inactive in initiating SV40 DNA replication in
an assay consisting of purified enzymes [26,27].
We then reproduced the absence of JCV species specifi-
city using the monopolymerase DNA replication system,
which makes use of purified enzymes only and allows
measurement of deoxyribonucleotide incorporation after
coupled initiation and elongation by DNA polymerase
a-primase [11]. Figure 4 shows that SV40 DNA replication
in this system is clearly dependent upon DNA polymerase
a-primase being of human origin (panel A) but that this is
not the case for JCV (panel B). This shows that the ability of
murine DNA polymerase a-primase to replicate JCV DNA
is not dependent upon factors other than the replication
proteins used in this assay. However, overall deoxynucleo-
tide incorporation is less efficient than by human DNA
polymerase a-primase as shown above with the cell-free

assay (Fig. 1), which suggests that steps in JCV DNA
replication subsequent to primer formation may be slightly
inhibited by the murine enzyme complex.
SV40 TAg confers species specificity to JCV origin
dependent DNA replication
It has been reported that SV40 TAg is capable of supporting
JCV DNA replication both in vivo and in vitro [32,33,47].
Therefore, we asked whether substitution of SV40 TAg for
JCV TAg would render JCV DNA replication species-
specific with regard to the nature of the DNA polymerase
a-primase complex catalysing the reaction, as is the case
Fig. 2. DNA synthesis products of the SV40
and JCV DNA replication systems. Murine
FM3A cell extracts depleted of DNA poly-
merase a-primase were supplemented with 1.0
DNApolymeraseunitsofH4orM4ornot
supplemented (–Pol). One-quarter of the
DNA synthesis products were analysed for
complete DNA replication by digestion with
EcoRI and DpnI (even numbered lanes). In
parallel, the products were linearized with
EcoRI (odd numbered lanes). The positions
of linearized template DNAs are indicated
by arrows.
Fig. 3. Autoradiogram of an in vitro JCV DNA replication initiation
assay with 0.2 and 0.4 units of primase of either human (H4) or murine
(M4) DNA polymerase a-primase complexes. Specific primase activities
were determined beforehand with a primase assay on poly (dT). Lanes
1 and 2, control reaction with DNA polymerase a-primase lacking
TAg or vice versa; lanes 3 and 4, 0.2 U and 0.4 U of human; lanes 5

and 6, 0.2 U and 0.4 U of murine DNA polymerase a-primase. The
approximate sizes of the reaction products are indicated on the right in
nucleotides (nt).
Ó FEBS 2003 Initiation of DNA replication (Eur. J. Biochem. 270) 2033
with SV40 DNA replication. Figure 5 shows that murine
DNA polymerase a-primase is indeed incapable of replica-
ting JCV DNA when the replication complex contains
SV40 TAg (columns 2–3). The reciprocal experiment to
investigate whether JCV TAg would relieve the species
specificity of SV40 DNA replication is not feasible as JCV
TAg is incapable of supporting SV40 DNA replication
[32,33].
Discussion
It has been firmly established that the species specificity of
lytic infection by the polyomaviruses SV40 and PyV is
determined at the level of DNA replication both in vivo and
in vitro by the nature of the host DNA polymerase
a-primase complex [2,19,21–27]. Here we report that the
closely related JC virus does not show such a strict
specificity in its DNA replication in vitro. Although murine
DNA polymerase a-primase is approximately 50% less
efficient than is its human counterpart in the replication of
JCV DNA in our assays (Figs 1–5), nucleotide incorpor-
ation by this complex is significantly above the values
determined in the SV40 DNA replication systems (Figs 1,2
and 4 [21,26,27]). In the purified initiation system of JCV
DNA replication, murine DNA polymerase a-primase can
catalyse primer formation at the JCV origin (Fig. 3), a
reaction the murine complex does not support in the SV40
system [22,26,27].

Importantly, we show that nucleotide incorporation in
the JCV system by murine DNA polymerase a-primase is
dependent upon the JCV DNA replication origin (Fig. 1B)
and results in DpnI-resistant products (Fig. 2), indicating
that it is due to bona fide DNA replication and not a
consequence of Ôfilling inÕ of gaps or other short patch repair
events. The fact that we observe a complete round of
plasmid replication in murine cell extracts indicates that
other essential replication proteins, such as DNA poly-
merases d and e, proliferating cell nuclear antigen (PCNA),
replication factor C (RF-C), topoisomerase I and DNA
ligase are not responsible for JCV species specificity in vitro.
Our murine FM3A cell extracts contain relatively low levels
of endogenous RPA and are therefore supplemented with
human RPA. However, if this is left out we nevertheless
observe significant, albeit overall less efficient, incorporation
by both murine and human DNA polymerase a-primase
(data not shown) indicating that RPA also is not a species-
specific factor.
In apparent contradiction of our results, Feigenbaum
et al. [8] showed that cultured nonpermissive hamster glial
cells were unable to replicate transfected JCV DNA. Their
observation and our data could be reconciled if the murine
but not the hamster cellular DNA replication machinery
were permissive for JCV DNA replication. We consider this
unlikely. A more likely reason for the discrepancy between
Feigenbaum’s data and our own is the difference in the
ÔstateÕ of the DNA in the two assays. Transfected DNA will
become associated with histones to form chromatin in the
cell nucleus whereas our in vitro assays are carried out with

naked plasmid DNA. Nucleosomes may interfere with the
initiation of replication and evidence exists that the binding
of transcription factors to sites in the regulatory regions
adjacent to the replication origin of the SV40 chromosome
may play a role in relieving such nucleosome repression [48–
52]. In vivo JCV DNA replication shows a greater depend-
ency on such flanking regions than does SV40 [33]. It could
be that JCV species specificity is governed by a host factor
such as nuclear factor I (NF-I) required to facilitate
initiation of replication in vivo but not in vitro, perhaps by
alleviating nucleosome repression or by assisting in origin
unwinding under conditions of altered template DNA
superhelicity [50]. The findings that JCV DNA replication is
stimulated by NF-I in vivo but not in vitro support this
explanation [50]. This view is consistent with the knowledge
that introducing an SV40 sequence into the JCV genome
Fig. 4. SV40 and JCV DNA replication using
the monopolymerase system. The SV40 system
contains SV40 TAg and DNA with the SV40
replication origin (A); the JCV system con-
tains JCV TAg and DNA with the JCV rep-
lication origin (B). DNA polymerase (0.25 and
0.5 U) of the indicated DNA polymerase
a-primase complexes were added (H4, human,
columns 2–3; M4, murine, columns 4–5; –Pol,
none, panels (A) and (B), column 1). Enzyme
activities were determined beforehand with a
DNA polymerase assay on activated calf
thymus DNA. Standard deviations from three
experimentsareindicatedaserrorbars.The

experiments in (A) and (B) were performed in
parallel.
2034 R. W. P. Smith and H. P. Nasheuer (Eur. J. Biochem. 270) Ó FEBS 2003
can extend the host range of JCV replication in vivo [52].
Alternatively, rodent, but not human, chromatin might
contain a nondiffusable factor that inhibits JCV TAg-
dependent DNA replication or the phosphorylation of JCV
TAg is different in human and mouse cells interfering with
the replication activity in vivo but not with the purified
baculovirus-expressed protein. This explanation is consis-
tent with findings that specific residues of SV TAg must be
phosphorylated whereas other may not be [17].
In summary, the available evidence strongly suggests
that, although DNA replication is the species-specific
process common to JCV, SV40 and PyV, different host
factors in each case ultimately determine the restriction of
virus propagation to a particular host. For SV40 and PyV
these are, respectively, the p180 and p48 subunits of DNA
polymerase a-primase in initiation of DNA replication
[22–24,26,27,53]. For JCV a different level of control
appears to be in operation although we cannot rule out
that the lower initiation efficiency of murine DNA
polymerase a-primase is at least in part involved when
compounded by other factors not present in our assays.
This view is supported by the recent finding that the JC
virus receptor is widely distributed on cells which contrasts
with the cellular restriction of virus propagation [54].
Factors involved in this regulation might be cellular
proteins such as puralpha, p53, or alternative splicing
products of viral proteins [51,55,56].

Substitution of SV40 TAg for that of JCV inhibits the
formation of an active initiation complex with murine DNA
polymerase a-primase (Fig. 5), mimicking the molecular
basis of SV40 host specificity. We recently showed that
species specificity of SV40 DNA replication is probably
the consequence of a failure of SV40 TAg to undergo a
productive functional interaction with C-terminal elements
of the murine p180 subunit of DNA polymerase a-primase
[26,53]. Our results imply that JCV TAg is not, or is to a
much lesser extent, inhibited in these interactions. SV40
and JCV TAg share 72% sequence identity with most
nonhomology towards the C-termini of the proteins [31].
The cell-free in vitro JCV DNA replication system would be
useful in determining which regions of SV40 TAg are
responsible for its species–specific interactions with host
DNA polymerase a, for instance by studying the activity of
chimeric TAg polypeptides derived in part from SV40 and
in part from JCV sequences.
Acknowledgements
We thank J. Fuchs and A. Schneider for technical assistance. This work
was financially supported by the Deutsche Forschungsgemeinschaft
(Na190/12 and Na190/13-1) and the EC (CT970125). The IMB is a
Gottfried-Wilhelm-Leibniz-Institut and financially supported by the
federal government and the Land Thu
¨
ringen.
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Fig. 5. Cell-free DNA replication assays in murine FM3A cell extracts
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polymerase a-primase complexes (human & murine) were tested in
parallel experiments for their ability to support DNA replication of a

plasmid carrying the JCV origin. Columns 2 and 3, SV40 Tag with
human and murine DNA polymerase a-primase, respectively; columns
4 and 5, JCV Tag with human and murine DNA polymerase a-pri-
mase, respectively. DNA polymerase (0.5 U) of the indicated DNA
polymerase a-primase complexes were added. Values were corrected
for background incorporation in the absence of added DNA poly-
merase a-primase (see Fig. 1). Column 1, control with H4 and plasmid
DNA but lacking TAg.
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