Somatic Celland MolecularGenetics, Vol. 20, No. 3, 1994,pp. 147-152
Electron Microscopic Analysis of In Vitro Replication Products of
8, a Mammalian Origin Enriched Sequence
ors
Christopher E. Pearson, Awatef Shihab-EI-Deen, Gerald B. Price,
and Maria Zannis-Hadjopoulos
McGill Cancer Centre, Departmentof Medicine, McGill University,3655Drummond Street,
Montreal, QuebecH3G 1Y6, Canada
Received 17 February1994---Final31 March 1994
Abstract--Electron microscopy was used to map the initiation site of ors 8 DNA replication in
vitro in a system that is capable of initiating and supporting one round of semiconservative
replication of cloned mammaliim DNA origin-enriched sequences (ors). Using unique restriction
sites in ors 8 plasmid DNA, we have mapped the replication bubble within the monkey DNA
sequence. In addition to site-specific initiation within the ors, the results also indicate bidirectional
replication.
INTRODUCTION
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. The observed synthesis was
semiconservative and appeared to be bidirectional (1). Here, we report the analysis of in
vitro replication products of ors plasmids and
mapping of the replication bubble by electron microscopy (EM).
We recently developed an in vitro
replication system that is capable of initiating
and supporting the semiconservative replication of cloned mammaiian origin-enriched
sequences (ors) (1). Four plasmids containing monkey (CV-1) nascent ors (2-4), which
had been shown previously to replicate
autonomously in transfected CV-1, COS-7,
and HeLa cells (5), were also found capable
of replicating in the in vitro system that uses
HeLa cell extracts (1). De novo site-specific
MATERIALS AND METHODS
initiation of replication on plasmids required
the prese,nce of an ors sequence, soluble
In Vitro DNA Replication. Reactions
low-salt cytosolic extract, polyethylene gly- were performed, using as template either ors
col, a solution containing the four standard 8 plasmid DNA or the vector pBR322 DNA
deoxyribonucleoside triphosphates, and an alone (Fig. 1). The reactions were essentially
ATP regenerating system. Replication of the as described previously (1) with the following
ors plasmids was not inhibited by ddTTP, an changes: no radioactive precursor nucleoinhibitor of DNA polymerase (3 and % and tides were used in the reaction, and all D N A
was sensitive to aphidicolin indicating that precipitations in ethanol were carried out at
DNA polymerase a and/or ~ was responsible -20~ overnight, in the absence of carrier
147
0740-7750/94/0500-0147507.00/0 ~ 1994PlenumPublishingCorporation
P e a r s o n et al.
148
j0
Pstl ~
(36o8,/
/
[
visualized using a Phillips 410 electron
microscope.
~
(561)
RESULTS AND DISCUSSION
ORS 8
plasmid
\^\
]~) 0.482kb
Fig, 1. Map of ors 8 plasmid. The solid line represents
the pBR322 vector (4.363 kb) DNA sequences; the box
indicates the ors 8 sequence (0.483 kb) cloned in the
NruI site of pBR322. The PstI (nucleotide 3608) and
SphI (nucleotide 561) restriction sites are indicated. The
map shown is drawn to scale.
tRNA. The in vitro replication reactions
were carried out for either 30 or 60 min at
30~ and the reaction products were precipitated and deproteinized as described previously (1). The samples were then treated
with 4 mg/ml of DNase-free RNase A
(Boehringer Mannheim) for 10 min at 37~
and this reaction was terminated by the
addition of one volume of 1% SDS, 1 mM
EDTA; proteinase K (BRL) was then added
to a final concentration of 200 mg/ml.
Following incubation of 1 h at 37~ the
samples were extracted once with phenol,
twice with ether, and precipitated with
ethanol, as before (1). After resuspension,
the DNAs were dialyzed against 10 mM Tris
hydrochloride (pH 8.0), 1 mM EDTA. When
restriction enzymes were used, digestions
were performed on deproteinized products
immediately before treatment with RNase.
All restriction enzyme digests were performed as specified by the suppliers.
Electron Microscopy. The products of
the in vitro replication reactions were spread
for EM analysis by the formamide method of
Davis et al. (6). The DNA was spread on
copper grids, shadowed with platinum, and
Mapping o f In Vitro Initiation Site. To
determine the site in the ors 8 plasmid (4.8
kb; Fig. 1) at which in vitro DNA synthesis
initiated, we digested the in vitro replication
products with appropriate restriction endonucleases (PstI or SphI), and analyzed them
by electron microscopy. For convenient reference points, the replication products were
linearized with either PstI, which cleaves the
ors 8 plasmid approximately 2.6 kb downstream from the ors 8 sequence or with SphI,
which cleaves the plasmid approximately 0.4
kb upstream from the ors 8 sequence (Fig. 1).
Since both these enzymes cut the ors 8
plasmid DNA at a unique site, the expected
molecules should consist of a mixture of full
length linears (i.e., digestion products of
either completely replicated or unreplicated
molecules) and of linearized replication
intermediates (i.e., linear molecules with
either a replication bubble or with doublefork structures, depending on whether the
restriction site fell within or outside the
replication bubble).
EM screening of grids with DNA spreads
of reactions (30- and 60-minute incubations)
that used pBR322 as the template did not
reveal any replication intermediates (Table
1), confirming previous results that were
obtained in transient replication assays in
vivo (2) and in vitro (1). In contrast, EM
examination of undigested ors 8 plasmid
DNA products, after 60 rain of replication in
vitro, revealed several structures other than
input supercoiled or relaxed circular DNA
molecules, such as theta structures (Fig. 2a)
and intertwined catenated dimers (Fig. 2b),
which are common late intermediates in the
replication of circular DNAs (7, 8). These
structures were observed at a frequency of
5.4% and 3.2% for 30- and 60-min incubations, respectively (Table 1). Only molecules
EM Analysis o f ors 8 Replication In Vitro
149
Table 1. EM Analysis of In Vitro Replication Products
Number of molecules
Plasmid
Ors 8
pBR322
Incubation
time (rain)
Enzyme
treatment
Total
counted
With
bubbles
With
forks
PstI
PstI
SphI
SphI
184
124
300
264
333
240
>500
> 500
10 (5.4) a
4 (3.2)
7 (2.3)
12 (4.5)
9 (2.7)
1 (0.04)
0
0
4 (1.3)
3 (1.1)
10 (3.0)
11 (4.6)
0
0
30
60
30
60
30
60
3O
6O
~Numbers in parentheses indicate percentages.
Finally, in preparations of in vitro
of the expected correct size (4.8 kb for ors 8
plasmid and 4.3 kb for pBR322) were scored. replication products of ors 8 plasmid DNA
Preparations of in vitro DNA replica- that were digested with SphI, in addition to
tion products that were digested with PstI, linear molecules, molecules with doublecontained internal replication bubbles (Fig. forked structures (Fig. 2f) were observed at a
2c-e) at a frequency of 2.3% and 4.5% for frequency of 3.0% and 4.6% for the 30-min
30- and 60-min incubations, respectively and 60-rain incubations, respectively (Table
(Table 1). The location of each replication 1). The site of initiation was mapped as
bubble on the plasmid molecule was mapped above and, again, in all molecules with
by measuring with a planimeter the lengths double forks that were analyzed, it mapped
of the unreplicated strands flanking the within the ors 8 sequence (data not shown).
bubble with reference to the location of the Also observed in these preparations, albeit at
restriction site on the plasmid map (Fig. 3). a lower frequency, were molecules with small
The measurements were consistent with the internal bubbles (data not shown), as would
mapping of all replication bubbles to the ors be expected from digestion of replication
8 sequence, indicating that initiation of intermediates in which the bubble has not
replication occurred within ors 8. Also proceeded beyond the SphI site. Such molobserved in these preparations were replicat- ecules were observed with a frequency of
ing molecules with forked structures (both 2.7% and 0.04% for the 30-min and 60-min
double and single), as would be expected incubations, respectively (Table 1). Again,
from the digestion by PstI of replication the site of initiation was mapped as above
intermediates, in which the replication bubble and, in all the molecules with bubbles that
had proceeded beyond the PstI site. These were analyzed, it mapped within the ors 8
molecules occurred at a frequency of 1.3% portion of the plasmid.
and 1.1% for the 30-min and 60-min incubaThe replication products observed in
tions, respectively (Table 1). The site of this study with ors 8 are similar to those
initiation was mapped by measuring the observed for SV40 replication both in vivo
relative lengths of the nascent strands to the (9) and in vitro (10), suggesting similar
unrepticated portion of the molecule (Fig. 3). mechanisms of replication for these two
As before, in all the molecules analyzed, the DNAs. The fact t h a t the relative lengths of
initiation site mapped within the ors 8 the unreplicated strands were proportional
sequence.
to the bubble size within all molecules
150
Pearson et al.
Fig. 2. EM of ors 8 in vitro replication products. In vitro replication reactions were carried out and purified as
described in the text. After 60 rain, the DNA replication products were spread for EM without further treatment (a
and b), after linearization with PstI (c-e), or linearization with Sphl (f). The bar represents 0.25 vim.
EM Analysis of ors 8 Replication In Vitro
151
ors
8
% replicated
36
10
54
35
~
52
10
40
50
10.2
25
40
35
40
58
2
38
52
10
32
51
18
34
57.5
8.5
35
61.1
3.9
36
44
20
40
40
20 - -
:!:!:!:!:!:!:!:i!:i!i~ii~i
-
Fig. 3. Mapping by EM of ors 8 in vitro replication products digested with PstI. In vitro DNA replication reactions,
using ors 8 plasmid DNA as template, were carried out as described in the text. After digestion with PstI, the DNA
was spread for analysis by EM. Molecules with replication bubbles were photographed and measured with a
planimeter, as described in the text. The top line indicates the ors 8 plasmid DNA linearized at the PstI site; the
position of the ors 8 insert within the plasmid is indicated (black box). The numbers represent the portion of the
plasmid occupied by the ors 8 insert (10%) and by the vector (pBR322) sequences (36% upstream and 54%
downstream o f ors 8, respectively). Molecules with replication bubbles are drawn to scale. The numbers underneath
the lines indicate the percent of the unreplicated molecule that lies on either side of the bubble. The percent of the
replicated molecule is shown in the right-hand column. The shaded region indicates the location of the ors 8 insert.
containing internal bubbles and that all
bubbles invariably m a p p e d within the o r s 8
sequence (Fig. 3) suggests that replication
proceeds bidirectionally. T h e same observations were true with regard to the linear
molecules containing double forks.
T h e frequency of replicating molecules
observed in vitro is in a g r e e m e n t with those
previously r e p o r t e d for the E M analysis of in
vivo transient episomal replication of o r s 8
and o r s 1 2 plasmid D N A (5). F u r t h e r m o r e ,
the estimated n u m b e r of molecules with
evidence of replication, and the extent of
replication, corresponds favorably to previous estimates of replication efficiencies in
vitro, based on m e a s u r e m e n t s of the picomoles of [ c x - 32p]dCTP i n c o r p o r a t e d into
o r s 8 plasmid D N A (0.4 pmol) after 1 h of
replication in vitro (1). In this study, we
observed by E M analysis that, after 1 h of
replication in vitro, the majority of replicating molecules were < 4 0 % replicated. If we
assume, for p u r p o s e s of comparison, that all
molecules undergoing replication were approximately 40% replicated, the observed
incorporation of radioactive p r e c u r s o r nucleotide would c o r r e s p o n d to 1% of the input
plasmid being replicated. Thus, the percentage of molecules with replication intermediates observed by E M (3.2%) c o m p a r e s
favorably to the estimated minimal n u m b e r
of molecules u n d e r g o i n g replication based
u p o n incorporation of radioactive p r e c u r s o r
(1%). It should be n o t e d that only the
molecules that are actually u n d e r g o i n g replication at the time of p r e p a r a t i o n for E M
152
analysis are scored by this method, while the
molecules that are either fully replicated or
unreplicated are not. Thus EM analysis
provides an underestimate of in vitro replicated molecules.
In summary, the results presented in this
study suggest that, during in vitro replication
of o r s 8 plasmids, initiation occurs within the
ors and proceeds bidirectionally. This is in
agreement with our previous observations
regarding ors plasmid replication, both in
vitro (1) and in vivo (5), and suggests that the
basic mechanism of replication in the crude,
reconstituted, cell-free system (in vitro) is
similar to that operating during transient
episomal replication of transfected plasmids
(in vivo).
Some systems have not detected specific
initiation sites, either in autonomously replicating plasmids (11-14) or in animal cells
(15, 16). However, the replication origins of
the D H F R amplicon (17), the c - m y c gene
(18, 19), the adenosine deaminase gene (20,
21), the immunoglobulin heavy chain gene
(22) and enhancer region (23), and an
autonomously replicating clone, 343 (24),
among others (reviewed in 25), exhibit
specific initiation sites and have been shown
to serve as chromosomal origins of replication. Although, it is difficult to reconcile
these two different views on sequence-specific
initiation, in most cases initiation of replication
seems to occur at specific sites (25).
ACKNOWLEDGMENTS
We would like to thank Max Zollinger
(Universit6 de Montr6al), for his expert
technical assistance with the platinum shadowing. C.E.P. is recipient of a Freedman
studentship and a Faculty of Medicine
Internal studentship. Dr. Shihab-EI-Deen
was on academic leave from the Faculty of
Medicine, Kuwait University. This work was
supported by grants from the Medical Research Council (MRC) of Canada (MT7965) to M.Z.-H. and from NSERC to G.B.P.
Pearson et al.
and M.Z.-H. M.Z.-H. is recipient of an MRC
Scientist Award.
LITERATURE CITED
1. Pearson, C.E., Frappier, L., and Zannis-Hadjopoulos,
M. (1991). Biochim. Biophys.Acta 1090:,156-166.
2. Zannis-Hadjopoulos, M., Kaufmann, G., and Martin, R.G. (1984)../. MoL Biol. 179:577-586.
3. Zannis-Hadjopoulos, M., Kaufmann, G., Wang,
S.-S., Hesse, J., and Martin, R.G. (1985). Mol. Cell.
Biol. 5:1621-1629.
4. Rao, B.S., Zannis-Hadjopoulos, M., Price, G.B.,
Reitman, M., and Martin, R.G. (1990). Gene
87:233-242.
5. Frappier, L., and Zannis-Hadjopoulos, M. (1987).
Proc. Natl. Acad. Sci. U.S.A. 84:6668-6672.
6. Davis, R., Simon, M., and Davidson, N. (1971).
Methods Enzymol. 21:413~28.
7. Sundin, O., and Varshavsky, A. (1980). Cell
21:103-114.
8. Wasserman, S.A., and Cozzarelli, N.R. (1986).
Science 232:951-960.
9. Martin, R.G., and Setlow, V.P. (1980). Cell
20:381-391.
10. Li, J.J., and Kelly, T.J. (1985). Mol. Cell. Biol.
5:1238-1246.
11. Heinzel, S.S., Krysan, P.J., Tran, C.T., and Calos,
M.P. (1901). Mol. Cell. Biol. 11:2263-2271.
12. Caddie, M.S., and Calos, M.P. (1992). Nucleic
Acids Res. 20:5971-5978.
13. Krysan, P.J., Smith, J.G., and Calos, M.P. (1993).
Mol. Cell. Biol. 13:2688-2696.
14. Krysan, P.J., and Calos, M.P. (1991). Mol. Cell.
Biol. 11:1464-1472.
15. Vaughn, J.P., Dijkwel, P.A., and Hamlin, J.L.
(1990). Cell 61:1075-1087.
16. Dijkwel, P.A., and Hamlin, J.L. (1992). Mol. CelL
Biol. 12:3715-3722.
17. Burhans, W.C., Vassilev, L.T., Caddie, M.S.,
Heintz, N.H., :ind DePamphilis, M.L. (1990). Cell
62:955-965.
18. McWhinney, C., and Leffak, M. (1990). Nucleic
Acids Res. 18:1233-1242.
19. Vassilev, L.T., and Johnson, E.M. (1990). MoL
Cell. Biol. 10:4899~904.
20. Carroll, S.M., DeRose, M.L., Kohlman, J.L.,
Nonet, G.H., Kelly, R.E., and Wahl, G.M. (1993).
Mol. Cell. Biol. 13:2971-2981.
21. Virta-Pearlman, V.J., Gunaratne, P.H., and
Chinault, C.A. (1993). Mol. Cell. Biol. 13:59315942.
22. Iguchi-Ariga, S.M.M., Ogawa, N., and Ariga, H.
(1993). Biochim. Biophys. Acta 1172:73-81.
23. Ariizumi, K., Wang, Z., and Tucker, P.W. (1993).
Proc. Natl. Acad. Sci. U.S.A. 90:3695-3699.
24. Wu, C., Zannis-Hadjopoulos, M., and Price, G.B.
(1993). Biochim. Biophys. Acta 1174:258-266.
25. Burhans, W.C., and Huberman, J.A. (1994).
Science 263:639-640.