156
Biochimica et Biophysica Acta, 1090 (1991) 156-166
© 19~1 Elsevier Science Publishers B.V. All rights reserved 0167-4781/91/$03.50
ADONIS 016747819100213B
BBAEXP 92295
Plasmids bearing mammalian DNA-replication origin-enriched
(ors) fragments initiate semiconservative replication
in a cell-free system
C h r i s t o p h e r E. P e a r s o n , Lori F r a p p i e r * a n d M a r i a Z a n n i s - H a d j o p o u l o s
McGill Cancer Centre, Department of Medicine, McGill Unit'ersity, Montreal (Canada)
(Received 18 January 1991)
(Revised manuscript received 21 June 1991)
Key word~: !n v.~r3 repl. "a~io¢; Cell-free sy~!e-~; R,'pl[cation origin; Scmiconscrvative replication; Initiation; DNA replication
Four plasmids containing monkey (C'V-I) origin-enriched sequences (ors), which we have previously shown to
replicate autonomously in CV-l, COS-7 and HeLa cells (Fcappier and Zannis-Hadjopoulos (1987) Prec. Natl. Acad.
Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo
site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low.salt cytosolie
extract, poly(ethylene glycol), a solution containing the four standard deaxyribouucleoside triphosphates and an
ATP regeaerati~ system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both
in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to
obtain closed circular supercoiled molecules. Replicotive intermediates migrating slower than form I1 and topoisomers migrating between forms !1 and I were also included among the replication products. Replication of the o ~
plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase tB and y, and was sensitive to aphidicolin
indicating that DNA polymerase a a n d / o r /~ was responsible for DNA synthesis. Origin mapping experiments
showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at
ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of
nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be
bidirectional.
lntrnductiou
The mechanisms regulating the replication of many
prokaryotic and viral DNAs have been extensively investigated mainly due to the availability of cell-free
systems for these DNAs. Such systems are lacking for
mammalian DNA. Chromosomes of eukaryotes initiate
DNA replication at multiple sites and considerable
evidence from yeast [1,2], Ustilago [3] and mammalian
[4-9] systems indicates that specific origins of replication exist in eukaryotes as they do in prokaryotes.
Based on their ability to serve as replication origins for
* Present address: Department of Microbiology, Cornell University
Medical College, New York, NY 10011, U.S.A.
Correspondence: M. Zannis-Hadjopoulos, McGill Cancer Centre,
Department of Medicine, McGill University, 3655 Drummond Street,
Montreal, Quebec, Canada H3G IY6.
plasmid DI~IAs replicating episomally, several autonomously replicating sequences (ars) have been isolated from Saccharomycescerevisiae, which are thought
to represent yeast chromosomal origins of replication
[1,2,10]. DNA fragments with ars activity have also
been isolated from human and mouse DNA [11-13].
Using a different approach, we isolated and cloned
from synchronized CV-1 (monkey) cells, early-replicating DNA sequences, which should contain origins of
replication [14,15]. 25 of these origin-rich sequences
(ors) have been characterized in detail and their properties [16,17], as well as primary sequence [16,18], have
been described. We have reported that several of these
enable transfected pBR322/ors recombinant plasmids
to replicate autonomously in monkey (CV-1 and COS-7)
and human (HeLa) cells [6,16]. This replication initiated within the ors and was carried out in a controlled
and semiconservative manner characteristic of mammalian replicons.
The development of cell-free replication systems
suitable for viral replication has provided ;mportant
157
information about the replication process in those systems [19-30]. Cell-free systems using extracts of monkey and human cells, that can replicate SV40 DNA in
an origin-dependent manner, have been described
[25,26,31-35], as have mouse cell extracts that can
replicate DNA containing the polyoma virus origin
[36]. Initiation of replication at SV40 or polyoma origins in these in vitro systems requires that the cell
extracts be supplemented with large quantities of the
virus encoded large T antigen (Tag), although synthesis
has been observed in its absence [35]. These in vitro
replication systems seem to mimic in vivo replication
processes and therefore have been useful for the identification of putative host replication proteins [26,3740] and their subsequent functional characterization
[24,27,30,39,41-47].
In this paper, we describe a mammalian in vitro
replication system capable of supporting the replication of plasmids that contain four monkey ors (ors 3, 8,
9 and 12) previously shown to replicate autonomously
in vivo [6]. Extracts prepared from HeLa cells are
capable of supporting the replication of the ors plasmids in vitro, in reaction conditions similar to those
described by Decker et al. [48], but excluding SV40 T
antigen. The in vitro replication of the ors plasmids is
sensitive to the action of aphidicolin but not of
dideoxythymidine triphosphate (ddTrP), and the initiation of replication is ors-specific, bidirectional arid
semi-conservative.
potassium acetate, 0.5 mM MgC! 2, 0.5 mM DTI') at
4 o C. The cells were collected by scraping with a rubbet policeman and lysed in a Dounce homogenizer
(four passes of pestle B). Nuclei were removed by
centrifugation at 1200 x g for 5 min and were used to
prepare the nuclear extract. The supernatant was spun
at 100000 x g for 1 h in a Beckman type 50 Ti rotor
and this supernatant (cytosol) was aliquoted and stored
at -70°C. Ten 15-cm diameter plates of HeLa cells
routinely yielded 2.5 ml of cytosol.
The nuclear pellet was suspended in 1.3 ml of byputonic buffer plus 500 mM KoAc and extracted for 90
min on ice with occasional vortexing. This extract was
then spun in a Beckman SW50.1 rotor at 300000 × g
for 1 h, and the resulting supernatant (nuclear) extract
was stored at -70°C.
Extracts made from HeLa $3 cells, adapted for
suspension culture were made essentially as above with
slight modifications [51]. Briefly, cells growing in midlog phase ((4-5). 105 cells/ml) were harvested by centrifugation (600 × g for 15 min). All procedures were
carried out at 4°C. The washing procedure entailed the
re-suspension of the ceils in the appropriate buffer to a
final concentration of 5" 106 cells/ml. Upon re-suspension in hypotonic buffer (as above) at 7.10 7
cells/ml, the cells were incubated in wet ice for 15 min
then lysed and nuclei collected as above. Nuclear pellets were re-suspended in 2.5 x vol. of the pellet in
hypotonic buffer plus 500 mM KOAc. All subsequent
steps were identical to those described above.
Materials and Methods
Cells and plasmids
HeLa cells (monolayers) were cultured ia D,'n,o,'co's
minimal essential medium (MEM) containing 5% fetal
calf serum. HeLa $3 cells were maintained in suspension in Eagle's minimal essential medium for suspension (SMEM) with 10% fetal calf serum, pBR322 and
pBR/ors plasmids were propagated in Escherichia coli
HB101 as previously described [6]. To eliminate template activity due to heterogenous DNA segments that
may arise in plasmid preparations from E. coli [49], all
plasmid DNAs were isolated by the alkaline lysis
method [50] without the use of chloramphenicol.
pBR/ors plasmids are comprised of CV-I monkey
DNA sequences inserted into the Nrul site of pBR322
[14,17].
Preparation of cell extracts
Extracts from log phase HeLa cell monolayers were
prepared as described by Decker et al. [48]. Cell monolayers were washed twice with isotonic buffer (20 mM
Tris-HCl (pH 7.4), 137 mM NaCI, 5 mM KCI, 1 mM
CaCI2, 0.5 mM MgCI2) plus 250 mM sucrose, then
with hypotonic buffer (20 mM Hepes (pH 7.8), 5 mM
Replication reactions
In vitro replication was carried out in a 50 /tl
reaction using 15/zl of cytosol, 250 ng of plasmid DNA
and 8/zl of nuclear extract, unless otherwise indicated.
Reactions also contained a final concentration of 45
mM Hepes (pH 7.8), 5 mM MgC! 2, 0.4 mM DT]', 1
mM EGTA, 60 mM sucrose, 240 mM ethylene glycol,
5% poly(ethylene glycol) (M r 12000, Fluka), 6 mM
phosphoenolp~uvate, 0.3 U pyruvate kinase (Boehringer-Mannbeim), 2 mM ATP, 100 mM ,.~dch CTP,
GTP, UTP, dATP, dGTP and dTrP, 10/zM dCTP and
approx. 10 #Ci of [a-32p]dcrP. When aphidicolin
(Boehringer-Mannheim) was included, it was added to
a final concentration of 30 ~tM. Reactions were incubated at 3 0 " C for 1 h unless otherwise indicated.
Reactions were terminated by the addition of 1 voi. of
1% SDS, 30 mM EDTA, and the DNA was purified
and concentrated as described by Decker et al. [48],
unless otherwise indicated. For calculating the pmol of
[ot-32p]dC'['P incorporated at various time intervals
during the in vitro reaction, the purified DNA products
were precipitated with 10% trichioracetic acid (TCA),
filtered through G F / C f'dters, dried and counted by
liquid scintillation as described in Maniatis et al. [52].
158
Semiconservath'e replication
Restriction digests and gel electrophoresis
Replication reactions were done as described above
except 5-bromodeoxyuridine triphosphate (BrdUTP)
(Sigma) was used in place of d'lTP and was added
fresh to reaction mixes, to a final concentration of 100
/~M. To produce non-substituted (LL) replication
products parallel reactions were performed as usual
using d'ITP. After the second ethanol precipitation
DNAs were run on G-50 sephadex (Nick columns,
Pharmacia) to eliminate free nucleotides, then re-precipitated, digested with Pstl or Alul (BRL) for 2 h at
3"PC, and analyzed by equilibrium centrifugation on
CsC! [53]. Under neutral conditions the DNA products
were loaded directly onto gradients with an initial
refractive index of 1.403. Under alkaline conditions the
DNA products were denatured for 15 min at room
temperature in 0.15 M NaOH, 0.1 mM EDTA; CsCI
(dissolved in 50 mM NaOH, 3 mM EDTA) was then
added to a final refractive index of 1.405. Gradients
were spun in a Vti80 rotor for 14 h at 65K and
followed by 2 h at 70K. Approx. 30 fractions were
collected from the bottom and densities were calculated from refractive indices that were recorded using a
refractometer (Fisher Scientific). Aliquots of 50 /~l
were taken from each fraction and counted Cerenkov
in a scintillation counter.
To assess the amount of replication of the plasmids
in each in vitro reaction, purified DNA was subjected
to electrophoresis on 1% agarose gels. The gels were
then dried and the incorporation of [a-32p]dCTP into
pBR/ors plasmids was compared to that of pBR322 by
autoradiography. For the Dpnl assay [54], the purified
in vitro replication products were digested with 2 U of
Dpni (BRI.) at 37°C for 6 h. The completeness of
digestion was verified by inclusion of A DNA in the
reaction mixture. To map the initiation site of the
plasmids that were replicated in vitro, the DNA was
digested to completion with Sau3A (BRL), or doubly
digested with Bgll (BRL) and BstNl (New England
Biolabs) and the resulting restriction fragments were
fractionated in 4% or 5% polyacrylamide gels. The gels
were then dried, autoradiographed and subjected to
densitometry scanning (LKB Bromma 2202 UItrascan,
Laser Densitomer) in order to quantitate the relative
radioactivity in each fragment.
l-P~3227
a b c d
l•
f
ors3
7
g
h i
["
j
k
Results
Replication of ors DNA templates in citro
We tested the ability of HeLa cell extracts to support DNA replication of cloned monkey DNA se-
ors8
7
I m n
r"
ors9
7
o P q r s
I- ors12
7
t u v w x
I1.~
II I'~"
Fig. 1. Time-courseof ors plasmidreplicationin vitro, pBR322and pBR/ors 3, 8, 9 and 12 plasmidDNAs(250 ng/reaction)wereincubatedat
30°(2in reactionn.ixturescontainingHeLa cellextracts for 5 rain(lanese, i, o, t), 10 min(lanesa, f, k, p, u), 20 min (b, g, I, q, v), 30 min (c, h, m,
r, w) and 60 min (d, i, n, s, x). The DNAswere purified,concentrated,and one third (83 ng) of each reactionwas subjectedto electrophoresis in
1% aSarose.Supercoiled(D, relaxedcircular(!1)and linear(lid formsof plasmidDNAsare indicated.
159
0.5-
-~ 0.4~0.3.
0.2.
0
5
t0
15 20 25 30 35 40 45 50 55 60
TIME (minutes)
Fig. 2. Picomolcs of [a-32p]dCTP incorporation as a function of
time. One third of each reaction (83 ng) described in FiB. 1 was
precipitated with 10% TCA [37] and radioactivity counted by scintillation counter, w, pBI~; , , ors 3; I I , "~rs8; • , o r s 9; e, orsl2.
quences containing origins of replication (ors). Four
monkey ors (ors 3, 8, 9 and 12) cloned in pBR322
[14,17,18], that have been shown to replicate autonomously when transfected in mammalian cells [6]
were used as templates, as was the vector alone, in a
ceil-free system using HeLa cellular extracts. Essentially no radioactive precursor was incorpolated into
pBR322 DNA, whereas the incorporation into the four
pBR/ors plasmids was consistently higher (Figs. 1 and
2). A limited amount of incorporation in pBR322 was
observed sometimes and was found to be due to repair
(see below). Control experiments where exogenous
DNA was not included in in vitro reactions yielded no
products (data not shown). Futhermore, clones containing random genomic sequences of similar size to
the ors clones were negative when assayed in the in
vitro sy.~em (data not shown) as they were also when
transfected into HeLa cells [55]. Extracts from monolayers, were as active in replication assays as were
extracts from suspension cells.
The pi-oducts of the in vitro reaction included relaxed circular (form II) linear (form Ill) and supercoiled (form l) plasmid DNAs (Fig. 1). The detection
of a ladder of bands migrating between forms II and l
indicated the presence of a series of topoisomeric
molecules with supercoils. Approx. 30-50% of the relaxed circular molecules became supercoiled when
electrophoresis was carried out in the presence of 6
p.M etnidium bromide (data not shown) indicating that
the form II plasmids are composed of both relaxed
dosed-circular and nicked-circular molecules. The
presence of high salt nuclear extracts in the in vitro
reactions was necessary for the formation of completely supcrcoiled (form I) plasmid DNA (Fig. 1).
Although nuclear extracts have been shown to inhibit
the replication of SV40 in vitro, they are necessary for
the formation of negatively supercoiled (form 1) SV40
molecules [48,25]. In our system too, the nuclear ex-
tract had both a weak super-coiling activity, as indicated by the low proportion of form 1 plasmid DNA
that was produced by comparison to form li, and an
inhibitory activity; when the nuclear extract was excluded from the reactions, the incorporation of nucleotides into the replicating plasmids increased approx. 8-fold but no form I DNA was detectable (data
not shown). In general, we have found that topoisomerase activity varies from one extract preparation to
another. In addition to supercoiled (form 1) and relaxed circular (form II) DNAs, we routinely observed
for all four ors plasmids material migrating slower than
form !!, indicative of replicative intermediates (RI) and
catenated dimers.
A time-course of the in vitro replication of ors 3, 8,
9 and 12 plasmids demonstrated a gradual increase in
the incorporation of precursor nucleotides into the
plasmid DNAs over a period of 60 rain (Figs. 1 and 2).
Initiation events occurred within the first 5-10 rain of
incubation, and incorporation reached a plateau by 90
rain (data not shown); by 20 min, some supercoiled
topoisomers were also detectable. In contras,, the rate
of incorporation of nucleotides into pBR322 DNA was
5- to 25-fold lower than that for the ors plasmids by 60
rain (Fig. 2). The amount of pnol incorporated ranged
from 0.3 to 1.254 pmol of dNMPs per 250 ng of DNA
in 60 rain or approx. (2-7)- 10 -3 of the value that has
been reported for SV40 systems [25,33-35,43,56]. Similar incorporation profiles were obtained when in vitro
DNA products were linearized with Pstl, run on 1%
agarose and full length template (form IID molecules
were excised and counted, as described by Guo et al.
[56] (data not shown).
The rapid increase of all replication products following 10 min incubation (Fig. 1, lanes g-i, I-n, q-s, v-x)
suggests a time lag of synthesis of less than 10 min. in
in vitro replication of SV40 a time lag of 10-15 rain has
been observed [20,25,29,32,39,57]; however, recently it
was reported that the time lag for SV40 in vitro replication could be reduced by pre-incubation of the SV40
Tag with a fraction of cellular extract prior to the
addition of the template [57].
Resistance of replication products to digestion by Dpnl
and Mbol
The in vitro replication products of the ors plasmid
DNAs were resistant to digestion by Dpnl (Fig. 3),
which only cleaves DNA that is metbylated on both
strands, suggesting that these DNAs are either hemimetbylated or unmcthylated and had therefore undergone at least one round of replication in the in vitro
system. Their additional resistance to digestion by Mbo l
(data not shown), which cleaves only unmetbylated
DNA, indicated that the majority of each ors DNA
plasmid underwent only one round of replication in
vitro.
160
I-aPBRb-II - : rs 3b'-11-:rs8~l
b
I_:rs9
and reinforce the previous results that suggest that
DNA synthesis in the cell-free reactions involves the
replicative DNA polymerases a a n d / o r 8. Incorporation into pBR322 DNA was inhibited approx. 50% by
ddTl'P but not completely eliminated; repair-type synthesis, however, cannot be ruled out, as no evidence of
specific replication for this plasmid was obtained in our
system (see below).
_ors12..
b I
i)"J I a
4 II
q III
ql
Evidence that the in vitro system yielded products of
sewac"onservative replication
In order to further verify that the DNA products
obtained in the in vitro reactions were the result of
semiconservative DNA replication, reactions were performed as described above in the presence of BrdUTP
in place of dTI'P and analyzed by isopycnic centrifuga-
pBR
I
--
ors3
ors8
ors9
orsl2
--
--
--
--
4-
4-
4-
4-
Fig. 3. Dpnl resistance. Replicationreactionswere performed and
treated as descn'bedin Fig. 1 excludingnuclearextracts. Reactions
products were digested for 6-7 h at 37°C, then analysed on 1%
aBarese gels. Lanes a undigested in vitro DNA products; lanes b,
Dlml digestedin vitroDNA products.
'--"
Effectof aphidicolin
The incorporatio~ of precursor nucleotides into ors
plasmids was sensitive to 30 ~tM aphidicolin (Fig. 4),
but not completely inhibited by it. A titration of aphidicolin concentrations ranging from 3.75/~M to 60/~M
showed a significant inhibition of incorporation (greater
than 80%) at the lowest concentration and a gradual
increase of inhibition through to 60 /~M (data not
shown). These results suggest that DNA polymerases a
a n d / o r ~ were largely responsible for the in vitro
nucleotide incorporation, as aphidicolin is a specific
inhibitor of these polymerases [58-60].
Effect of ddTTP on in vitro DNA replication
To determine whether any of the labelling of the
plasmid DNAs in the in vitro reactions might be due to
repair-type synthesis carried out by DNA polymerases
or 3,, reactions were performed in the presence of
2',3'-dideoxythymidine triphosphate (ddTrP), an inhibitor of these poiymerases but not of polymerase a
[48]. The results (Fig. 5) show that even very high
concentrations of ddTTP (200 ~tM) failed to inhibit the
incorporation of radioactive precursor into ors 3, 8, 9
and 12 DNAs. These results indicate that incorporation of precursor nuclcotides into the ors plasmids in
vitro is not carried out by DNA polymerases/3 or 3',
• II
•11!
•1
!
Fig. 4. Effect of aphidicolin on in vitro reactions, pBR322 and
pBR/ors 3, 8, 9 and 12 plasmidDNAs were incubatedin reaction
mixtures containingHeLa cytosulicextracts in the presence (t-) or
absence ( - ) of 30 tiM aphidicolin,as described in Materials and
Methods. Aftera 60 rain incubationat 30~ the DNAswere purified,
concentrated and subjectedto electrophoresis in a 1% agamse gel.
Relaxed circular (I!) and linear (lid forms of plasmid DNAs are
indicated.
161
tion. Under alkaline conditions the in vitro products of
all ors plasmids yielded clear distinct peaks at the
density expected of single-stranded substituted (H)
molecules, as can be seen in sample gradients of ors 3
and ors 9 (Fig. 6A and B, respectively). Similar profiles
were obtained for ors 8 and 12 (data not shown).
BrdUTP substitution was incomplete (75-80%), in the
in vitro reactions, possibly due to endogenous amounts
of dTTP present in cellular extracts as has also been
previously reported by others [33]. The similarity in
density shifts observed both when the rci~lication products were digested by A/ul, which generates 16 or
more fragments, and by Pstl, which generates full
length linear DNAs, is suggestive of BrdUTP incorporation due to semiconsevative replication as opposed to
repair. If the incorporation were due to repair the
amount of BrdUTP incorporated would not be sufficient to cause a density shift, especially in short fragments [61], such as those generated by A/ul digestion.
Under neutral conditions the products for all four ors
plasmids banded near the densities expected for singly
substituted (HL) and unsubstituted (LL) molecules
(data not shown). The absence of doubly substituted
(HH) molecules indicates once more that in the in vitro
system initiation of replication of the four ors occurs
,----pER
ddTTP (uM)
0
Fig. 5. Effect of d~rl'fP on ors plasmid D N A
,,
SO 300 O
3
only once, with virtually no additional rounds of replication taking place. These results are similar to those
we obtained in vivo [6] with the same ors plasmids. In
contrast, the overall incorporation of precusor in
pBR322 vector was 20-30-fold lower than that in the
ors-containing plasmids in both neutral and alkaline
gradients and the profiles obtained were low and broad,
suggesting that its incorporation is due to repair type
synthesis [61,69], as our data above also indicate. Profiles of semiconsevative replication similar to ours have
also been obtained using a wide range of eukaryotic
and prokaryotic DNA templates in cell-free DNA
replication systems of Xozopus eggs [62,63], in microinjected Xenopus eggs [62-64], and in viral in vitro
replication systems [25,33]. Similar results were obtained also in runoff replication assays using Avian
DNA [66], plasmids containing the chromosomal origin
of the human c-myc gene [67] or the amplified cellular
dihydrofolate reductase origin of replication [68].
Mapping o f the in vitro initiation site on ors ~lasmid
Finally, to determine whether the replication of ors
plasmids initiated within the ors, the three ors plasmid
DNAs that replicated with the highest efficiency in the
cell-free system (ors 3, 8 and 9) were incubated in the
.
so 2oo o
8
so
, r--9
-
5too o so
.,
24m o
1 2 ....
so2oo
replicatio.,pBR322 and ors.3, 8, 9 and 12 plameuklswere im:~baled in reactioa mixtures containing
H©La¢ytosolicextractsand 0, 50 and 200 #M ddi-IV for 60 rain. Rcactiomp m d ~ ~
~
~ ~
m F~ 1.
162
i
L
H
~o = 1.8664
I
10+
p= 17730
-
E
(3.
C~I
0
5
20 t
H
10
15
20
25
'O= 1"7795
;j
\
olA: "./ k:0
5
t0
15
20
25
30
FRACTION
Fig. 6. lsopycnic centrifugation in alkaline gradients. Reactions were
performed as described in Materials an," Methods using both cytosolic and nuclear extracts. One third of the reaction (83 ng) was
subjected to restriction enzyme digestion with Pstl for ors 3 and
pBR322; or with A/ul for ors 9, and banded in alkaline CsCI
gradients. Profiles for ors 3 (A) and ors 9 (B) are shown together
with pBR322 as the control. Arrows denote the positions of substituted (H) DNA, where 5-bromodeoxyuridine triphosphate was used
in place of d T I P ( o , pBR322; - , ors 3; ¢,, ors 9) and of the
positions of non-substituted (L) products where replication reactions
were performed as usual with dTTP (11, pBR322; zx, ors 3; <>, ors
9). The densities ( g / m l ) at peak fractions are indicated.
(A)
~
(B)
j~lO0
j lO0
ise
in vitro replication mixture for 15 rain at 30°C, and
subsequently digested with S a u 3 A and fractionated on
a polyacrylamide gel (Fig. 7, insets). During this incubation period the ratio of initiated, but incompletely
replicated, molecules to completely replicated
molecules should be high, and therefore the region at
and around the origin of replication should be preferentially labelled. The incorporation of [a-32p]dCTP
into each restriction fragment was quantitated by scanning several exposures of the autoradiographs with a
densitometer, and from these values the specific radioactivity of each fragment was calculated. The plots
of relative DNA synthesis per base pair for each plasmid DNA fragment of ors 3 (Fig. 7A), ors 8 (Fig. 7B)
and ors 9 (Fig. 7(2) indicate that, in each case, the
ors-containing fragments (c and h for ors 3; c and g for
ors 8; and c and g for ors 9) were preferentially
replicated in a 15 rain reaction. In contrast, when
pBR322 DNA was used as a template no such sitespecific incorporation was seen (Fig. 7(2), suggesting
once more that the low-level nucleotide incorporation
into this plasmid is probably due to repair-type synthesis. A plot of the relative DNA synthesis per base pair
of DNA for each fragment of pBR322 DNA generated
by S a u 3 A digestion is not shown, since the bands from
all but the longest fragment of pBR322 were too faint
to be detected by densitometer scanning, even in longer
autoradiographic exposures. These data suggest that
replication of the ors plasmids starts within the ors
sequences and proceeds bidirectionally. In the case of
ors 3 and ors 9 it appears that initiation might occur
,,
~54D
'g'U'
C 'lt~l
~rsa
]
'
b
'f'
i
•
plasmtd Fra~m~mts
C
.
ii
•
c
on
•
"
Plasmid F~--qmlnts
Fig~ 7. Mapping of the origin of replication on ors plasmids, ors 3 (A), ors 8 (B), ors 9 (C) or pBR322 (C) plasmids were replicated in HeLa
¢ytosolic extracts for 15 rain at 30°C, then were digested with Sau3A and fractionated on a 5% acrylamide gel. The incorix~ation of [3zp]dCT]P
into each restriction fragment of the plasmids (a to i) was determined by densitometer tracings of the autorndiolFaphs shown above each graph,
and the relative DNA synthesis per base pair of DNA was determined for each fragment. Several film exposures were analysed to ensure a linear
range in film response. The plasmid DNA fragments containing ors sequences are indicated on the x-axis of each graph.
163
-~,o.
o,,
~
o ~ 8 gee~me (bp)
Fig. 8. Time-course mapping of the origin of replication on ors 8
plasmid, ors 8 was incubatedas in Fig. I for 5, 15, (sofidshading)25,
(cross-hatchedshading)35, (hatched shading)and 60, (white) rain at
300C, the reaction products were digested with Bgll and BstNl,
fractionated on a 4% acrylamidegel and subjected to autoradiography. The incorporationof [32PklCTP into each restriction fragment of the plasmidswas determined by densitometertracingsof the
autoradiographs; several film exposures were analysed to ensure a
linear range in film response.The area under each peak was divided
by the number of base pairs (bp) in that fragment to compensatefor
differences in fragment length, and the relative amount of DNA
synthesis/bp in each fragmentwas normalizedto the fragmentwith
the highest specific radioactivity,which was defined as 100%. The
ors 8 plasmid DNA fragments generated by Bgll/BstNl digestion
are indicatedon the x-axisand the ors sequencesare indicatedby a
solid line.
nearer the 3' end of the ors sequence and that replication proceeds at a slightly higher efficiency in the 3' to
5' direction from the origin. Previous studies of in vitro
initiation of SV40 D N A have demonstrated, in general,
bidirectional replication from the origin [33,35,43,48],
although these data do not exclude the possibility of
site-specific initiation reactions followed by replication
proceeding unidirectionally on individual molecules
[35,43]. Similar experiments were performed with ors
3, 8 and 9 using incubation times of 20 and 60 min of in
vitro replication, and the products were digested with
Sau3A, electrophoresed, autoradiographed and analysed by densitometry. The data (not shown) again show
that by 20 min the initiation of replication has occurred
in the ors and proceeds bidirectionaily throughout the
whole genome by 60 min.
A more detailed time-course (5, 15, 25, 35 and 60
min incubation) of in vitro replication was performed
for ors 8 and the replication products from the various
times of incubation were doubly digested with BglI
and BstNl yielding 10 fragments ranging from 13 to
1060 bp. Again, the relative amount of D N A synthesis
per bp was plotted for each D N A fragment (Fig. 8).
Histogram analysis of the genomic distribution of radioactive precursor incorporation as a function of time
shows that the ors-containing fragments G and E have
the highest specific activity at 15 min and at all subsequent time points. D N A synthesis progressed bidirectionally from the ors fragments and spread throughout
the entire genome as the time-course proceeded. At 5
min the amount of synthesis was not sufficient to
produce visible bands upon Bgll/BstNl digestion, even
after at~toradiographic exposures of 6 to 20-fold higher
than those required for the other time points. These
results are in agreement with those shown in Fig. 1.
The results show that at 60 min incubation there is still
preferential incorporation (initiation events occuring)
in the ors fragments; the amount of radiolabel per
base pair decreases in both directions from the ors,
again indicating bidirectional replication from the ors.
The same densitometric readings were also plotted
with respect to the G + C content of each fragment
and yielded similar graphs (data not shown).
Discussion
We have presented evidence that pBR322 derivative
plasmids containing monkey ors sequences are able to
initiate in a cell-free system using log-phase HeLa cell
extracts. When reactions were carried out in the presence of aphidicolin to inhibit DNA polymerases a and
8, we found that approx. 10% of ors plasmid DNA
labeling was aphidicolin-resistant. Using an SV40 in
vitro replication system, Decker et al. [48] have shown
that DNA synthesis in the region of the SV40 origin is
much less sensitive to aphidicolin than DNA synthesis
further away from the origin, resulting in the accumulation of early replicative intermediates in the presence
of aphidicolin. Thus, the partial sensitivity of ors plasmid DNAs to aphidicolin could be due to the initiation
of RNA-primed D N A synthesis by HeLa cell primaseDNA-polymerase a complex [48], as human DNA primase is resistant to aphidicolin [70,71]. The sensitivity
to aphidicolin and resistance to d d T r P of ors plasmid
replication in vitro indicates that, like cellular DNA
replication, DNA synthesis in the cell-free system is
carried out by D N A polymerases a a n d / o r &
In our system, the in vitro replication products migrated as relaxed circular and linear pla.qnids, with
only a limited number of supercoiled molecules being
detected. In reactions using cytosol alone (data not
shown), some topoisomers were evident indicating that
the cytosolic extract had some limited ability to induce
negative supercoiling, as has also been reported by
others [25,72]. It is possible that the cellular extracts
are low in topoisomerase (I a n d / o r II) activity which
has been demonstrated to be essential for both facilitated and complete replication of the SV40 virus in
vitro [74]. Low activity of topoisomerase could account
for the incomplete RI (catenated forms) and the low
amounts of form I DNA. Other in vitro replication
systems require supplementation of cellular extracts
with topoisomeras~s) to enhance as well as obtain full
monumeric (form I and II) molecules [27,51,73,74]. As
in other in vitro replication systems [32,48,75] poly(ethylene glycol) stimulated replication initiation but appeared to inhibit production of form !. The latter could
164
be due to inhibition of one of the steps of replication
termination, and not of topoisomerase activity, as PEG
has been shown to increase the processivity of topoisomerase [76]. The stimulatory effect of PEG on in vitro
rcplication might be due to its ability to not only
stabilize replication protein complexes but also to stimulate many other reactions, such as association of ribosomal subunits, DNA h~;nding of polymerases, DNA
idnasing, and intermolecular DNA ligation (Ref. 77,
and references therein).
It has been suggested that production of forms II
and III may be due to increased susceptibility of plasmid DNAs to digestion by endonucleases [48]. Linear
reaction products have been reported in several cellfree replication systems [31,48,73], and are most likely
due to the presence of endonucleases in the cell extracts. Alternatively, it has been suggested that linear
structures may be real replicative intermediates that
are produced during segregation of two daughter
molecules, when both strands of the molecules are cut
transiently [31]. Varying degrees of success in achieving
complete circular plasmid replication in vitro have also
been reported in the various SV40 systems [25,35,48].
A detailed analysis of the replication products is beyond the scope of this report.
The ors containing plasmids 3, 8, 9, and 12 replicate
in vitro in a semiconservative manner, similar to that
observed in vivo [6]. The absence of detectable amounts
of doubly substituted molecules suggests that templates
undergo single initiations. These results are similar to
those obtained in other DNA replication systems using
either eukaryotic or prokaryotic DNA templates with
Xenopus cell extracts [62,63] or in whole Xenopus eggs
[65]. These results also reflect those found in viral
systems in similar experiments, namely that a small
proportion of template molecules actually initiate
replication in vitro and of those that do only a few [25],
if any [33] undergo multiple rounds of replication. In
vivo, SV40 can undergo multiple rounds of replication
in a single cell cycle [54,78,79], while mammalian origins of replication normally initiate only one round of
replication per cell cycle.
Origin mapping experiments, in which we analyzed
the pattern of labeling of restriction fragments early in
the replication reaction, as well as at several time
points throughout the reaction, indicate that replication of ors 3, ors 8 and ors 9 plasmids initiates within
the ors and proceeds bidirectionally. Although a limited amount of incorporation into pBR322 is sometimes seen, there is no evidence for site-specific initiation, suggesting that in this plasmid the incorporation
is due to repair-type synthesis.
It is functionally significant that four ors plasmids,
that were previously shown to replicate autonomously
in vivo (ors 3, 8, 9 and 12) are also capable of initiating
replication in the in vitro ::'-t~m. Transfection experi-
ments, in CV-1, COS-7 and HeLa cells, have shown
that ors plasmid replication is most efficient in HeLa
cells [6]. Similarly, the in vitro replication of ors plasmids using HeLa cell extracts was several fold more
efficient than that using equivalent extracts of CV-1 or
COS-7 cells (data not shown). Other laboratories have
reported differences in in vitro replication activities of
cell extract source [25,34,35,43,56]. In particular the
replication activity of cytosol fractions was found to be
higher in transformed human cell lines as were extracts
of SV40 infected cells compared to their non-infected
counterparts. The data presented here and previously
[6] suggest that HeLa cells may produce initiator proteins in higher concentrations than CV-1 or COS-7
cells.
Although the in vitro system that we have presented
is capable of initiating replication on monkey ors fragments, it is clearly not an optimal system for initiation
of mammalian origins. In SV40 in vitro replication
systems, efficient initiation at the SV40 origin requires
the addition of relatively large quantities of purified T
antigen [25,33,35,48], the viral initiator protein, although extensive in vitro synthesis of SV40 DNA in the
absence of Tag has been reported [35]. Initiator proteins which activate mammalian replication origins have
not yet been identified.
Additional ors-containing plasmids (ors 1, 10 and
11), that have been previously described [6,18,80], which
had not been found to replicate consistently in vivo at a
level detectable by the D/m I-resistance assay [6], were
found to replicate in vitro (data not shown). Since
replication of these plasmids in vivo was not asses~ed
by any methods other than the Dpnl-resistance assay,
it is possible that these plasmids undergo controlled
replication in vivo and that the level of replication was
too low to be consistently detected by the D p n l assay
[6]. Alternatively, the observed differences in the replication of these plasmids in vitro and in vivo may
indicate that the requirements for replication in the
cell-free system are more relaxed than those in the
intact cell or nucleus.
In vitro synthesis was dependent upon the presence
of an ors template and did not occur with clones of
random genomic DNA of similar size. The exact sequences within ors 3, 8, 9 and 12 that enable them to
act as replication origins, both in vitro [6] and in vitro,
are presently under investigation. Although these ors
do not share extensive sequence homologies, they do
share common features, such as AT-rich regions and
inverted repeat sequences [18,80], that may be important for origin function. With regard to the latter,
introduction of an anti-crucifo:m-DNA monocional antibody in synchronized and ~ermeabilized cells enhanced DNA replication 3- to ll-fold the normal level
[81-83]; similar effects have been obtained in vitro
using ors 3, 8, 9, and 12 (unpublished data). Recently,
165
b o t h in vivo a n d in vitro c r u c i f o r m e x t r u s i o n at t h e
origin o f r e p l i c a t i o n o f t h e s t a p h y l o c o c c a l p l a s m i d h a s
b e e n s h o w n to b e involved in initiation o f r e p l i c a t i o n
[84]. T h e f o r m a t i o n o f t h e s t a p h y l o c o c c a l c r u c i f o r m is
e n h a n c e d by t h e b i n d i n g o f its initiator p r o t e i n R e p C
[84].
T h e r e q u i r e m e n t o f a n ors s e q u e n c e in t h e cell-free
s y s t e m p r e s e n t e d h e r e , will b e useful for t h e fine
m a p p i n g o f t h e ors f u n c t i o n a l d o m a i n a n d s h o u l d
facilitate t h e i d e n t i f i c a t i o n o f s e q u e n c e s a n d s t r u c t u r e s
t h a t a r e i m p o r t a n t f o r t h e initiation o f m a m m a l i a n
D N A r e p l i c a t i o n , as well as o f r e p l i c a t i o n p r o t e i n s
involved in t h e r e g u l a t i o n o f c u k a r y o t i c D N A replication initiation e l o n g a t i o n a n d t e r m i n a t i o n .
Acknowledgments
T h i s w o r k was s u p p o r t e d by g r a n t s f r o m t h e M e d i c a l
Research Council (MRC) of Canada (MA-7965) and
T h e C a n c e r R e s e a r c h Society, Inc. L.F. was r e c i p i e n t
o f a n M R C s t u d e n t s h i p . W e t h a n k Mrs. L o r r a i n e W e l c h
for h e l p in t h e p r e p a r a t i o n o f t h e m a n u s c r i p t .
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