Reprinted from Nature, Vol. 332, No. 6164, pp, 546-548, 7 April 1988
Macmillan Magazines Ltd., 1988
A role for the nuclear
envelope in controlling DNA
replication within the cell cycle
J. Julian Blow & Ronald A. Laskey
CRC Molecular Embryology Research Group,
Department of Zoology, Downing Street, Cambridge CB2 3E1, UK
In eukaryotes the entire genome is replicated precisely once
in each cell cycle. No DNA is re-replicated until passage
through mitosis into the next S-phase. We have used a
cell-free DNA replication system from Xenopus eggs to
determine which mitotic changes permit DNA to re-replicate.
The system efficiently replicates sperm chromatin, but no
DNA is re-replicated in a single incubation1. This letter
shows that nuclei replicated in vitro are unable to rereplicate in fresh replication extract until they have passed
through mitosis. However, the only mitotic change which is
required to permit re-replication is nuclear envelope
permeabilization. This suggests a simple model for the
control of DNA replication in the cell cycle, whereby an
essential replication factor is unable to cross the nuclear
envelope but can only gain access to DNA when the nuclear
envelope breaks down at mitosis.
When sperm chromatin is incubated in homogenates of
Xenopus eggs it is assembled into normal interphase nuclei
surrounded by a nuclear envelope1-4 (Fig. la, b). When such
extracts are permitted to synthesize protein (>40 µg protein per
ml extract in 4 h), nuclei assembled in vitro subsequently
undergo the early mitotic changes of chromatin condensation
and nuclear envelope breakdown2 (Fig. lc, d).
In addition, DNA assembled into nuclei in vitro is replicated
efficientlyl,5-7. Bromodeoxyuridine density substitution shows
that only a single round of replication occurs in a single incubation1 in the presence or absence of protein synthesis (Fig. 2a).
The extract distinguishes replicated from unreplicated nuclei.
Nuclei replicated in the presence of cycloheximide, and then
transferred to fresh extract, do not re-replicate (Fig. 2h, filled
symbols and Fig. 3h). If protein synthesis is permitted, however,
replicated nuclei slowly acquire the ability to re-replicate in
fresh extract (Fig. 2b, open symbols and Fig. 3a). The ability
to re-replicate corresponds to the time at which chromatin
condensation and nuclear envelope breakdown occur ( Figs 1, 2).
To determine if nuclear envelope breakdown and chromatin
condensation are the changes that permit nuclei to re-replicate,
nuclei were replicated in the presence of cycloheximide and
then given various treatments before transfer to fresh extract
(Fig. 3c-f). Figure 3c shows the effect of treatment with
Maturation Promoting Factor (MPF). This is an activity found in
most mitotic or meiotic cells that can induce mitotic changes
in interphase nuclei9-11. Treatment with MPF, which causes
nuclear envelope breakdown and chromatin condensation, can
substitute for protein synthesis in allowing nuclei to re-replicate
when added to fresh extract (Fig, 3a-c).
Figure 3d-f shows that nuclear envelope permeabilization
alone is sufficient to allow re-replication. Thus when nuclei are
replicated in the presence of cycloheximide, and then treated
with agents that permeabilize the nuclear envelope, they can
re-replicate in fresh extract. Lysolecithin, a lecithin analogue
which inserts into lipid membranes and permeabilizes them,
permits 43% of the replicated DNA to re-replicate (Fig. 3d).
Melittin and phospholipase, which hydrolyses phospholipids in
the nuclear envelope, permit 27% of the replicated DNA to
re-replicate (Fig. 3e). Similarly, mechanical shear, such as
that caused by pelletting the nuclei in a fixed angle rotor,
permits replicated DNA to re-replicate (ref. 1 and data not
shown). However, degradative treatments that do not
permeabilize the nuclear envelope, such as phosphatase (Fig.
3f) and RNase (data not shown) do not allow re-replication.
When only a fraction of the template DNA re-replicates
Fig. 1 Protein synthesis permits mitotic changes in vitro. Sperm
chromatin (3 ng DNA per µ1) was incubated for 5 h in extract plus
(a, b) or minus (c, d) 100 µg ml-1 cycloheximide. Nuclei were
examined under Hoechst 33258 UV fluorescence (a, c) or phase
contrast optics (b, d). Scale bar 20 µm; all fields at same
magnification,
Methods. Extracts of Xenopus eggs were prepared by the method
described1 with the following modifications. Prior to crushing, eggs
were packed by centrifugation at 1,500 r.p.m. in an SW50 rotor
(Beckman) for 1 min at 4°C, and all excess buffer was removed.
Cytochalasin B 10 µg ml-1 was added to the extract after
crushing. Extract was frozen with 3% v/v glycerol.
(Fig. 3c-e) most nuclei re-replicate either fully or not at all (flow
cytometry data, not shown). Therefore nuclear envelope
permeabilization during mitosis is sufficient to permit nuclei to
re-replicate completely in the subsequent interphase.
We have previously discussed positive and negative regulatory
models for limiting DNA replication to a single round per cell
cycle12,13 but experiments performed during the course of this
work exclude aspects of each of them (data not shown). Instead
these results can be explained if an essential replication factor
is unable to cross the nuclear envelope during interphase and
can only gain access to DNA when the nuclear envelope breaks
down at mitosis. This model is outlined in Fig. 4. In Fig 4a this
Factor (Licensing Factor) is shown binding to chromatin during
mitosis or at the start of an incubation. Before DNA can replicate, it must be assembled into a nucleus with a complete nuclear
envelopel,4-6,14. In the model, no unbound Licensing Factor
remains free in the nucleoplasm: possibly because it is stable
in the nuclear environment only when bound to DNA, or
because its only route for inclusion in the nucleus is by binding
DNA, nascent nuclear envelope being very closely applied to
the surface of the chromatin4 (Fig. 4b). Only after the DNA is
assembled into a mature nucleus are replication forks initiated
throughout the nucleus at the sites of bound Licensing Factor
(Fig. 4c) Licensing Factor only supports a single initiation event
where it is bound to the DNA, and is inactivated by either
initiation or the passage of a replication fork (Fig. 4d). This
means that the entire genome is replicated precisely once, and
no re-replication can occur (Fig. 4e). Nuclei in G2 are unable
to re-replicate because Licensing Factor in the cytoplasm cannot
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Time of transfer (hours)
Fig. 2 The extract can distinguish replicated from unreplicated
DNA. a, Density substitution of sperm chromatin incubated in
vitro shows a single round of semi-conservative replication with or
without cycloheximide. b, Time course showing that replicated
DNA can be re-replicated an addition to fresh extract only in the
presence of protein synthesis.
Methods. a, Sperm chromatin (3 ng DNA per µl) was incubated
in egg extract with α32P-dATP and 0.4 mM BrdUTP for 5 h; DNA
was extracted and fractionated on CsCl density gradients. Density
shown by arrows: HH 1.79; HL 1.75; LL 1.71. (□), Control
(untreated) extract; (◆) extract with 100 µg ml-1 cycloheximide.
b, Sperm chromatin (3 ng DNA per µl) was incubated in 20 µl
extract with α32P-dATP, 0.5 mM BrdUTP, plus or minus 100
µg ml-1 cycloheximide, for various times, and then resuspended in 1
ml Buffer A (ref. 5). Nuclei were pelleted at 2,000 g for 2 min, and
resuspended in fresh extract containing 3H-dATP, 0.5 mM
BrdUTP, plus or minus 100 µg ml-1 cycloheximide. This extract was
incubated for 4 h, when DNA was isolated and fractionated on CsCl
density gradients1. (□), First and second extracts untreated (no
cycloheximide). (◼), First and second extracts contained 100 µg ml-1
cycloheximide. Either first (●), or second (○) extracts only
contained cycloheximide.
gain access to the DNA until the nuclear envelope is
permeabilized during mitosis. Thus this model can explain
why G2 nuclei in G11 G2 cell hybrids cannot re-replicate until
passage through mitosis15. Alternative models involving the
escape of a diffusible inhibitor at mitosis would have to be much
more complex than the one presented here.
Neither the Xenopus egg nor egg extract require specific DNA
sequences for DNA replication 1,6,13,16,17 , nuclear formation1,6,13,16,17 or for limiting DNA replication to a single
round per cell cycle16,20. The occasional re-replication of plasmid
DNA in incubations in vitro1 probably reflects the fragility of
pseudo-nuclei assembled from pure DNA in vitro. The Xenopus
embryo therefore differs from bovine papilloma viruses, which
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Fig. 4 Model for the control of DNA replication in the Xenopus
early embryo. a, Licensing factor (+) hinds to DNA. b, DNA is
assembled into nucleus. c, Initiation at licensed sites occurs coordinately throughout individual nuclei5. d, Licensing factor is inactivated by initiation or the passage of a replication fork. e, Fully
replicated DNA cannot re-replicate due to exclusion of licensing
factor from DNA by nuclear envelope. Breakdown of the nuclear
envelope during mitosis allows access of the Licensing Factor to
the DNA, to prepare it for DNA synthesis in the next cell cycle.
special cis-acting sequences to constrain viral
replication to a single round per cell cycle21,22.
Consistent with our model, G2 nuclei from HeLa cells must
be prepared using detergent to be capable of replication in the
first cell cycle after microinjection into Xenopus eggs23. But
many eukaryotic cells, such as yeast, do not undergo nuclear
envelope breakdown during mitosis. The model could still operate
in these organisms so long as nuclei become permeable to the
Licensing Factor during mitosis. The morphological changes of
the yeast nucleus during mitosis24 invite the speculation that the
model presented here may be applicable to all eukaryotic
cells.
require
Fig. 3 Nuclear envelope permeabilization between incubation in
two successive extracts allows re-replication of replicated nuclei
(seen as HH DNA). a, Nuclei replicated in the absence of cycloheximide. b-f, Nuclei replicated in the presence of cycloheximide
and treated with: b, mock treatment; c, maturation promoting
factor; d, lysolecithin; e, mellitin and phospholipase; f, phosphatase. Incubation conditions were as for Fig. 2b. The percentage of
32
P-labelled DNA in each fraction is given. The 3H content of
each fraction was also measured; this confirmed the identification
of the peaks as arrowed.
Methods. Panels a and b, nuclei were transferred as for Fig. 2b.
c, Extract was supplemented with 50% volume of MPF11 for 2 h
before nuclei were transferred as for Fig. 2b. d, Nuclei were pelleted as
for Fig. 2b, resuspended in 100 µg ml-1 lysolecithin (100 µl) for 10
min, diluted with 400 µl Buffer A plus 2% BSA, underlayered with
fresh extract, and transferred by centrifugation at 5,000 r,p.m. for 2
min in an SW50 rotor (Beckman). e, Nuclei pelleted as for Fig.
2b were resuspended in 100 µl Buffer A plus 50 µg ml-1
melittin, 50 µg ml-1 phospholipase A, 1 mM Ca2+,1 mM Mg2+
for 10 min, diluted with 400 µl Buffer A plus 2% BSA, 1 mM
EDTA, and transferred as in d. f, Nuclei pelleted as for Fig. 2b
were resuspended in 50 µ1 Buffer A pH 8.2 plus 25 units of calf
intestine alkaline phosphatase, diluted and transferred as in d.
3
We thank David Blow, Steve Dilworth, Colin Dingwall,
Richard Harland, Peter Lachman, Moira Sheehan and Jim Watson for their help. This work was funded by the Cancer Research
Campaign. J.J.B. is a Beit Memorial Junior Research Fellow.
Received 11 December 1987; accepted 26 February 1988.
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