 |
INTRODUCTION |
The initiation of DNA replication at the G1 to S phase
transition is a key regulatory step of the cell division cycle in
eukaryotic cells. Once DNA replication has initiated, control
mechanisms ensure that the entire genomic DNA is replicated precisely
once, and after completion, one replicated genome segregates to each of
the two daughter cells during mitosis (for reviews, see Refs. 1-6).
Cell fusion experiments in mammalian somatic cells established that
G1, but not G2 phase nuclei, initiate DNA
replication prematurely when exposed to an S phase cytosolic
environment (7). Key regulators of initiation were identified in
genetic and cytological experiments in vivo. Roles for
cyclin-dependent protein kinases (Cdks)1 and their
G1 and S phase-specific regulatory subunits cyclin D, E,
and A in inducing DNA replication have been documented (8-16). The
analysis of initiation of DNA replication in vivo was
recently complemented by a biochemical approach through the
establishment of cell-free systems from human and mammalian cells
(17-19). DNA replication is initiated in nuclei isolated from human
G1, but not G2 phase cells when incubated in S
phase cytosolic extract and S phase-specific nuclear factors. A
nuclear extract could be substituted by purified recombinant cyclins A
and E, complexed to their kinase partner Cdk2, to initiate DNA
replication, directly demonstrating functional roles for these
nuclear cyclin·Cdk complexes (17). These cyclin/Cdks were
essential, but not sufficient, as nuclei also required soluble factors
present in a cytosolic extract from S phase cells to initiate
replication (17).
For an assembly of replication-competent chromatin in eukaryotic
G1 phase nuclei, an evolutionarily conserved series of
molecular events is required involving the origin-recognition complex
(ORC), Cdc6 protein, and the mini-chromosome maintenance (MCM) proteins (reviewed in Refs. 2-4, 20, and 21). In mammalian cell-free systems,
competence to initiate DNA replication in S phase cytosol is observed
when template nuclei are prepared from late G1 phase cells
after release from a block either in mitosis (17) or quiescence (18),
followed by transit through early G1 phase in
vivo. Upon release from quiescence, competence of G1
phase nuclei to initiate in vitro coincides with maximum
expression of Cdc6 protein, and addition of recombinant Cdc6 protein
advances the onset of replication competence in vitro (18).
However, human cells undergoing mitotic proliferation contain Cdc6
protein at all stages of the cell cycle (22-24) and the state of DNA
replication competence therefore depends on other factors as well.
Late G1 phase nuclei from cells synchronized by release
from either mitosis or quiescence are relatively undefined
heterogeneous and dynamic populations as a result of cells passing the
state of competence at the time of preparation (17, 18). A significant step toward molecular and temporal dissection of the establishment of
DNA replication forks in human somatic cells would therefore be the
availability of defined populations of homogeneous template nuclei
reversibly arrested in a state of replication initiation competence. A
recently established cell-free system for the initiation of nuclear DNA
replication in the yeast Saccharomyces cerevisiae made use
of cells reversibly synchronized at defined points in the cell division
cycle (25). Template nuclei from yeast mutants blocked at defined
points in late G1 phase initiated DNA replication at high
percentages upon incubation in S phase nuclear extracts (25). Because
such cell cycle mutants are currently not available as synchronization
tools for human cells, I sought to achieve analogous arrest of human
cells in late G1 phase by chemical synchronization.
The plant amino acid mimosine is a versatile agent for blocking DNA
replication in proliferating eukaryotic somatic cells but the
literature on its mode of action is controversial. Depending on cell
type and concentration of mimosine, evidence for blocking both
initiation and elongation steps of DNA replication has been reported
(26-34). In the case of human cultured cells, however, this
controversy has been clarified because the different effects of
mimosine depend on its concentration in the cell culture medium during
treatment (34). At concentrations below 0.5 mM, mimosine interferes with elongation steps of DNA replication, and treatment results in populations of cells enriched in S phase after establishment of replication forks (34). In contrast, at concentrations of 0.5 mM and above, mimosine additionally prevents entry into S phase, resulting in a population of cells arrested in late
G1 phase, before establishment of active DNA replication
forks in vivo (34). Importantly, the late G1
phase arrest in vivo is fully reversible and cells enter S
phase upon removal of mimosine (27, 34).
In this paper, I characterize the ability of nuclei from
mimosine-arrested human cells to initiate DNA replication in human cell
extracts. Nuclei prepared from these cells efficiently initiate semiconservative DNA replication upon incubation in cytosolic extracts
from proliferating cells, even in the absence of a nuclear membrane.
Initiation depends on a cytosolic extract from proliferating cells and,
furthermore, on cyclin E/Cdk2 and/or cyclin A/Cdk2 activity which are
present in the nuclei from mimosine-arrested cells. The data suggest
that the competence of nuclei from mimosine-arrested cells to initiate
replication is characterized by the presence of the cell
cycle-regulated cyclin/Cdk2 proteins in cis and initiation is triggered by a different activity in trans.
| |
MATERIALS AND METHODS |
Cell Culture and Synchronization--
HeLa-S3 cells were
cultured as monolayers and synchronized in G1, S, and
G2 phase exactly as described (17). Synchronization in
mitosis was performed as described (35). Cells were arrested in late
G1 phase by adding 0.5 mM mimosine (Sigma) from
a 10 mM stock solution to proliferating cells for 24 h
(34).
Cell synchronization in interphase was determined by flow cytometry of
isolated nuclei. One million nuclei were directly stained with
propidium iodide (5 µg/ml in phosphate-buffered saline containing 0.4% Triton X-100) and analyzed by FACScan (Becton Dickinson) using
the Lysis II-software. Data are presented as histograms showing
relative DNA content (x axis) and cell number (y axis).
Preparation of Nuclei and Cell Extracts--
Nuclei from
mimosine-arrested cells were prepared by hypotonic treatment, followed
by Dounce homogenization and centrifugation exactly as described (34).
Concentrations of nuclei were determined with a hemocytometer. Nuclei
were stored at 
80 °C for up to 3 months without loss of DNA
replication competence. For permeabilization, nuclei were incubated in
0.1% Triton X-100 in SuNaSpBSA (250 mM sucrose, 75 mM NaCl, 0.5 mM spermine trihydrochloride, 0.15 mM spermidine tetrahydrochloride, 3% bovine serum albumin)
at 4 °C in a rotator for 20 min and washed two times in SuNaSpBSA
without Triton X-100.
Cytosolic and nuclear extracts from asynchronously proliferating and
synchronized cells were prepared exactly as described (17, 35). Protein
concentrations were determined with the Bio-Rad Protein assay using
bovine serum albumin as standard. Cytosolic extracts were frozen in
liquid nitrogen and stored up to 4 months at 80 °C without loss of
replication initiation activity.
DNA Synthesis Reactions and Analysis of Reaction
Products--
DNA replication initiation reactions (17) contained the
following components: HeLa cell cytosolic extract (100 µg of protein, unless indicated otherwise); a buffered mixture of rNTPs and dNTPs including either biotin-16-dUTP (Roche Molecular Biochemicals) or
[
-32P]dATP as tracers (17); and 2-5 × 105 nuclei from mimosine-arrested HeLa cells (34). The
final reaction volume of 50 µl was adjusted with replication buffer
(20 mM K-HEPES, pH 7.8, 100 mM potassium
acetate, 1 mM MgCl2, 0.1 mM
dithiothreitol). Where indicated, reactions were also supplemented with
S phase nuclear extract (50 µg of protein). Incubation time was
3 h, unless indicated otherwise.
The Cdk-inhibitors roscovitine and olomoucine (both Calbiochem) were
dissolved in dimethyl sulfoxide at 50 mM. When used, they
were added to replication reactions at the final concentrations specified in the figure legends. Control reactions contained an equivalent volume of dimethyl sulfoxide.
Recombinant cyclin A/Cdk2 and cyclin E/Cdk2 were prepared from SF9
cells infected with recombinant baculovirus expression vectors (gifts
of W. Krek, Friederich-Miescher-Institute, Basel). Purified human
cyclin B1/Cdc2 was a gift of M. Jackman (Wellcome/CRC Institute,
Cambridge) and cyclin D2/Cdk6 a gift of E. Laue and W. Zhang
(Department of Biochemistry, University of Cambridge).
For analysis by confocal fluorescence microscopy, nuclei were fixed and
spun onto coverslips. Total genomic and replicating DNA were visualized
and analyzed exactly as described in Refs. 18 and 34. Analysis of
radioactively labeled replication products by acid precipitation and by
density substitution were performed exactly as detailed before (Refs.
17, 18, and 34, and references therein).
Immunoblotting and Immunoprecipitation--
Immunoblots of
cytosolic and nuclear extracts from HeLa cells were performed
essentially as described (35). The following primary antibodies against
human proteins were used: anti-Cdk1, anti-cyclin A, and anti-cyclin B1
(all gifts from J. Pines, Wellcome/CRC Institute, Cambridge);
anti-cyclin D1 (ab171, AbCam); anti-Cdk2 (sc-163, Santa Cruz);
anti-Cdk4 (sc-260, Santa Cruz); anti-cyclin E (HE12, a gift of Ed
Harlow, Massachusetts General Hospital; sc-198, Santa Cruz).
Immunoprecipitation of cyclin E from human cell extracts was performed
with polyclonal antibody sc-198 (Santa Cruz) from a total of 150 µg
of extract protein diluted in 1 ml of phosphate-buffered saline (36).
The immunoprecipitate was washed in phosphate-buffered saline and
subsequently analyzed by Western blotting using monoclonal antibody HE12.
Depletion of Cdk1 and 2 from Cytosolic Extract--
Sepharose
beads coupled to human p9Cks1 protein (37) (a gift of M. Jackman, Wellcome/CRC Institute, Cambridge) were equilibrated and
washed three times in replication buffer and concentrated by gravity
sedimentation. Depletion of interphase cytosol was achieved by three
successive rounds of (i) adding a fifth volume of the
p9Cks1 beads, (ii) incubating the slurry for 20 min at
4 °C in a rotator, and (iii) removal of the beads by pelleting at
13,000 rpm in an Eppendorf 5415C centrifuge for 5 min. Mock depletions
were performed in parallel in the absence of
p9Cks1-Sepharose.
| |
RESULTS |
Reversible G1 Phase Arrest of Human Cells by
Mimosine--
Asynchronously proliferating human cells arrest in late
G1 phase when a 0.5 mM concentration of the
plant amino acid mimosine is added to the culture medium for 24 h
(34). Removal of mimosine from the culture medium in vivo
results in a synchronous entry into S phase in about 50-70% of the
cells (27, 34). Our previous work has established that a proportion of
late G1 phase nuclei prepared from cells released either
from mitosis (17) or quiescence (18) can serve as templates for
initiation of DNA replication upon incubation in human S phase
extracts. Therefore, I investigated whether the
mimosine-dependent reversible arrest point of human cells
before entry into S phase could be exploited for a preparation of more
homogeneous populations of competent and defined template nuclei for
efficient initiation of DNA replication in vitro.
Initiation of DNA Replication in S Phase Extracts--
Nuclei were
isolated from mimosine-arrested human cells and used as templates for
DNA replication reactions in vitro (Fig. 1). DNA synthesis was detected by
incorporation of biotin-dUTP into genomic DNA and confocal fluorescence
microscopy. Incubation of nuclei from mimosine-arrested cells in
elongation buffer resulted in about 10% of the nuclei incorporating
biotin-dUTP in vitro (Fig. 1A). These nuclei
represent a small proportion of contaminating true S phase nuclei
present in the preparation that continue semiconservative DNA
replication at sites established prior to preparation in
vivo (34). In contrast, addition of S phase cytosol to the
reaction supported DNA synthesis in about 50-55% of the nuclei (Fig.
1B). Because 10% of the nuclei had initiated DNA
replication in vivo before preparation (cf. Fig.
1A and Ref. 34), this result demonstrates that S phase
cytosol triggers initiation in about of 40-45% of the nuclei that had
been arrested by mimosine before establishment of DNA replication forks
in vivo. The percentage of nuclei synthesizing genomic DNA
depended on the amount of S phase cytosolic protein added and reached
saturation at 100 µg (Fig. 1C, columns 2, 5, and
6). Addition of nuclear extract from S phase cells also
increased the percentage of nuclei synthesizing DNA (Fig. 1C,
column 3). Addition of subsaturating amounts of both extracts
together increased the percentage of nuclei synthesizing DNA in
vitro in an additive fashion (Fig. 1C, column 4). The
maximum percentage of nuclei initiating DNA synthesis in
vitro was about 60 in most preparations, over and above the
contaminating proportion of S phase nuclei, corresponding to the
percentage of cells entering S phase in vivo upon removal of
mimosine.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Initiation of DNA synthesis in nuclei from
mimosine-arrested cells in S phase extracts. Nuclei were isolated
from cells arrested in late G1 phase by 0.5 mM
mimosine (34) and incubated in elongation buffer (A) or S
phase cytosol (B). Both incubations were in the presence of
nucleoside and deoxynucleoside triphosphates, including biotinylated
dUTP, and were analyzed by confocal fluorescence microscopy. Nuclear
DNA is visualized by propidium iodide (red signal) and DNA
replication by fluorescein-conjugated streptavidin (green
signal). Merged images are presented showing replicating nuclei in
yellow-green and non-replicating nuclei in red.
C, quantitation of the percentages of nuclei replicating in
cytosolic and nuclear extracts from S phase cells. Replication
reactions were performed in the presence of the indicated protein
amounts of cytosolic (c) and nuclear (n) extract.
The percentages of replicating nuclei were quantitated on microscopic
fields containing 500-800 nuclei. D-F, high magnification
analysis of a representative field of nuclei from mimosine-arrested
cells incubated in S phase cytosol. Visualization of the propidium red
channel (D, DNA), the fluorescein channel (E,
replication), and the merged image of both channels (F) is
presented. Scale bar, 10 µm.
|
|
The fluorescent signal of DNA synthesis in nuclei from
mimosine-arrested cells was not homogeneous within the nuclei (Fig. 1B). Replicating nuclei were therefore analyzed by confocal
microscopy at higher magnification (Fig. 1, D-F). Against
the background staining of nuclear DNA (Fig. 1, D and
F, red signal), a clear pattern of many very small discrete
intranuclear sites of DNA synthesis was detected (Fig. 1, E
and F, green signal). This pattern resembles the small
replication foci found in very early S phase, which are located in the
euchromatic regions of the nucleus (Fig. 1F; see Refs.
38-40, for reference). These data suggest that nuclei from
mimosine-arrested cells initiate DNA synthesis in S phase extracts
in vitro at sites used in early S phase in
vivo.
In other eukaryotic DNA replication initiation systems, access of
soluble initiation factors to the genomic DNA is regulated by the
integrity of the nuclear membrane (1, 18). Therefore, I asked next
whether removal of the nuclear membrane influences initiation of DNA
synthesis in this system. Nuclei from mimosine-arrested HeLa cells were
treated with 0.1% of the non-ionic detergent Triton X-100 to remove
the nuclear membrane and used as templates for DNA replication in S
phase cytosolic extracts (Fig. 2).
Nuclear membrane permeability was measured by exclusion of fluorescent dextran. About 90% of the template nuclei were permeable, and remained
permeable during the replication reaction in vitro (Fig. 2A). In comparison, without detergent treatment, typically
5-30% of the nuclei were permeable in a standard preparation by
Dounce homogenization (data not shown). Importantly, about 40% of the permeable nuclei initiated DNA synthesis upon addition of S phase cytosol (Fig. 2B), which is the same percentage as compared
with untreated nuclei (cf. Fig. 1C). Therefore,
access of soluble initiation activity from S phase cytosolic extract to
nuclei of mimosine-arrested cells is not influenced by the presence or
absence of an intact nuclear membrane. These data establish that the
terminal stages of initiation of human DNA synthesis in
vitro as observed in nuclei from mimosine-arrested cells do not
require the integrity of the nuclear membrane, consistent with
observations made in extracts from Xenopus eggs (41).
View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2.
Initiation of DNA replication in the absence
of a nuclear membrane. Nuclei from mimosine-arrested cells were
permeabilized with 0.1% Triton X-100. A, permeability of
the nuclear membrane of treated nuclei before and after a 3-h
replication reaction in vitro. The permeability of the
nuclear membrane was determined by exclusion of fluorescent dextran
using confocal microscopy. B, initiation of DNA synthesis in
permeable nuclei from mimosine-arrested cells in S phase cytosolic
extract. The percentages of nuclei replicating in the absence
(white column) and the presence of S phase cytosol
(black column) were quantitated as described in the legend
to Fig. 1.
|
|
These data so far do not allow a distinction between initiation of
semiconservative DNA replication or DNA repair synthesis in
vitro. Therefore, initiation of DNA synthesis was analyzed by
density substitution (Fig. 3). Nuclei
from mimosine-arrested cells synthesized only background amounts of DNA
upon incubation in elongation buffer (34), migrating between hemi- and
unsubstituted densities (Fig. 3A). In contrast, incubation
in S phase cytosol resulted in the synthesis of hemisubstituted DNA
products (Fig. 3B). During this 2-h incubation in S phase
cytosol, about 65 pmol of dNMP were incorporated into 105
replicating nuclei (data not shown), indicating that about 4-5% of
the genomic DNA was replicated. These data directly demonstrate that
semiconservative DNA replication is initiated in mimosine-arrested nuclei by a soluble activity present in cytosolic S phase extract.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Initiation of semiconservative DNA
replication in nuclei from mimosine-arrested cells in
vitro. Nuclei from mimosine-arrested HeLa cells were
incubated in elongation buffer (A) and in S phase cytosol
(B) in the presence of 5-bromodeoxy-UTP and
[-32P]dATP for 2 h. Reaction products were
analyzed by cesium chloride density equilibrium centrifugation as
specified under "Materials and Methods." The fraction numbers of
the gradients and the positions of DNA not substituted (light-light,
LL), hemisubstituted (heavy-light, HL), and fully
substituted with 5-bromodeoxyuridine (heavy-heavy, HH), as
determined by refractive indices, are marked.
|
|
These data raise the possibility that this soluble initiation activity
is inhibited by 0.5 mM mimosine in vivo, causing
cell-cycle arrest before onset of S phase (34) and allowing efficient
initiation of DNA replication in isolated nuclei upon incubation in S
phase extracts. In the next experiments, I therefore characterized the competence of cytosolic extracts from mimosine-arrested cells to allow
DNA replication in vitro.
Lack of DNA Replication Initiation Activity in Cytosolic Extract
from Mimosine-arrested Cells--
First, S phase nuclei were used as
control templates for elongation of DNA replication in vitro
(Fig. 4A). As demonstrated before (34), S phase nuclei elongate DNA replication at pre-existing replication forks in either elongation buffer or in S phase cytosol (Fig. 4A, white and black columns). Cytosolic
extract from mimosine-arrested cells allowed elongation in the same
percentage of S phase nuclei (Fig. 4A, gray column),
following the same incorporation kinetics as in S phase cytosol (data
not shown). Density substitution confirmed synthesis of hemisubstituted
DNA products in all of these three incubations (data not shown). These
results demonstrate that cytosol from mimosine-arrested cells
efficiently allows elongation of semiconservative DNA replication at
established active replication forks in vitro.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
Cytosol from mimosine-arrested cells lacks
initiation activity. A, elongation of DNA replication
by S phase nuclei in vitro. Template nuclei were prepared
from S phase HeLa cells and incubated in the absence of cytosolic
extracts (white column, labeled "buffer") or
in cytosolic extract (100 µg of protein) from mimosine-arrested cells
(gray column, "mim") and S phase cells
(black column, "S"). The percentage of nuclei
replicating was quantitated as specified in Fig. 1C.
B, DNA replication in nuclei from mimosine-arrested cells
in vitro. Nuclei from mimosine-arrested HeLa cells were
incubated in vitro as specified in panel A. C,
complementation of replication initiation activity in cytosol from
mimosine-arrested cells by G1 and S phase cytosolic
extracts. Nuclei from mimosine-arrested HeLa cells were incubated in
cytosolic extract from mimosine-arrested cells, that was supplemented
either with buffer only (white column, buffer),
or with cytosolic extract (50 µg of protein) from mimosine-arrested
(gray column, mim), S phase (black column, S),
and G1 phase cells (black column, G1).
|
|
In contrast, cytosol from mimosine-arrested cells allowed initiation of
DNA replication only in 15-20% of the nuclei from mimosine-arrested
cells (Fig. 4B, gray column), over and above the background
of S phase contaminants (Fig. 4B, white column). As S phase
cytosol triggered initiation in about 50% of these nuclei, the ability
of cytosol from mimosine-arrested cells was significantly, but not
entirely inhibited. This inhibition can either be explained by the
presence of dominant inhibitors of initiation (but not of elongation),
or the lack of soluble initiation activity in cytosol from
mimosine-arrested cells. To discriminate between these possibilities, I
supplemented cytosolic extract from mimosine-arrested cells with
cytosol from G1 and S phase cells (Fig. 4C).
Addition of subsaturating amounts of either G1 or S phase
cytosol fully restored the initiation of DNA replication (Fig.
4C), demonstrating that cytosol from mimosine-arrested cells lacks soluble initiation activity.
The data of Fig. 4C suggest that cytosol from G1
phase cells may also contain replication initiation activity. This
would point toward a requirement for a different soluble initiation activity than the S phase-specific cyclin·Cdk complexes observed before, using nuclei from cells synchronized in late G1
phase by release from mitosis or quiescence (17, 18). I therefore analyzed the initiation activity of cytosolic extracts from cells arrested at all stages of the cell division cycle in relation to the
presence of cyclin/Cdk proteins in these extracts.
Cell Cycle Specificity of Soluble DNA Replication Initiation
Activity--
HeLa cells were synchronized in early, mid, and late
G1 phase, and in S, G2, and M phase (Fig.
5). The presence of cell cycle-regulated cyclins and their kinase partners in cytosolic extracts from these cells was analyzed by Western blotting (Fig. 5A). Cyclin E
protein was not detectable in early G1 phase cytosol, but
was present in maximum amounts in mid and late G1 phase,
and in lower amounts in S, G2, and M phase cytosol. Small
amounts were also present in a salt extract from S phase nuclei (Fig.
5A, SN). Cyclin A protein was barely
detectable in cytosol from G1 phase cells, but was present
in large amounts in S and G2 phase cytosol. S phase nuclear
extract contained large amounts of cyclin A protein. Cyclin B1 protein
peaked in G2 cytosol and was barely detectable in the other
extracts. Cyclin D1 and the kinases Cdk1, Cdk2, and Cdk4 were clearly
detectable in cytosolic extracts from all stages of the cell cycle.
Additionally, Cdk2 was most abundant in S phase nuclear extract,
similar to cyclin A (Fig. 5A, SN).
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Cytosol from all phases of the cell cycle
allows initiation of DNA replication in nuclei from mimosine-arrested
cells. HeLa cells were synchronized in early G1
(G1e, 4 h post-release from
nocodazole-arrest), mid G1 (G1m,
6 h post-release), late G1
(G1l, 8 h post-release), S (2 h
post-release from thymidine block) and G2 phase (9 h
post-release from thymidine block), and in mitotic metaphase
(M, nocodazole-arrest) (17). Cytosolic extracts containing
cytoplasmic and nucleosolic proteins were prepared from these cells. A
high-salt extract from S phase nuclei (SN) was also
prepared. A, Western blot analysis of these extracts. For a
detection of cyclin E protein, it was first immunoprecipitated from 150 µg of total extract protein and immunoprecipitates were analyzed by
Western blotting. For the other proteins, identical amounts of each
extract (50 µg of protein) were directly analyzed by Western blotting
using antibodies against the indicated human cyclin/Cdk proteins (see
"Materials and Methods"). B, initiation of DNA synthesis
in nuclei from mimosine-arrested cells in these extracts. Reactions
contained identical amounts (100 µg) of the indicated cytosolic
extracts, and control reactions contained no cytosol (buffer,
white column) or both, S phase cytosol and 50 µg of protein of S
phase nuclear extract (S+SN). The percentages of
nuclei replicating were quantitated as described in the legend to Fig.
1.
|
|
Next, the DNA replication initiation activity of these extracts was
tested in vitro using nuclei from mimosine-arrested cells (Fig. 5B). Surprisingly, cytosolic extracts from all stages
of G1 phase cells triggered initiation of DNA replication
most efficiently in up to 60% of the nuclei. S and G2
phase cytosolic extracts were less active, triggering initiation in
about 25-40% of the nuclei and even cytosol of mitotic cells,
arrested in metaphase by nocodazole, triggered initiation in about 25%
of the nuclei. Initiation activity of S phase cytosol was stimulated by
addition of S phase nuclear extract to the maximal efficiency observed in late G1 phase cytosol alone (Fig. 5B).
These data clearly establish that the cytosolic DNA replication
initiation activity for nuclei from mimosine-arrested cells is not
strictly restricted to a particular phase of the cell cycle, however,
it peaks in G1 phase and is partially inhibited in mitosis. Most importantly, replication initiation activity does not simply correlate with the protein levels of the G1/S
phase-specific cyclin E·Cdk2 and cyclin A·Cdk2 complexes present in
these extracts.
These unexpected data raise two important questions, which I will
address in the remaining experiments successively. (i) Does cytosol
from untreated, asynchronously proliferating cells also trigger
initiation in nuclei from mimosine-arrested cells? If so, this would
provide an enormous simplification in the experimental protocol to
study the initiation of human DNA replication in vitro by
dismissing the requirement to synchronize cells for preparation of
initiating extracts. (ii) Is there a requirement for the
G1/S phase-specific cyclin·Cdk complexes to initiate DNA
replication in this system?
Interphase Cytosol Triggers Initiation of DNA Replication After a
Short Lag Period--
Nuclei from mimosine-arrested cells were
incubated in cytosolic extract from asynchronously proliferating cells
and the time course of DNA replication was followed in vitro
(Fig. 6). After an initial lag of 15 min,
cytosol from interphase cells triggered efficient initiation of
replication in about 30% of the nuclei during the following 45 min
(Fig. 6A, closed symbols). Then, the rate of initiation
dropped to about 7% of nuclei initiating per hour for the remaining
incubation. The amount of replicated DNA accumulated after an initial
lag for at least 2 h (Fig. 6B, closed symbols). In
control incubations in the absence of cytosolic extract, the background
percentage of nuclei replicating did not change over the 3-h incubation
period (Fig. 6A, open symbols) and only small amounts of DNA
were synthesized (Fig. 6B, open symbols). These data
demonstrate that interphase cytosol triggers initiation of DNA
replication in nuclei from mimosine-arrested cells efficiently after a
short lag in the initial hour of the in vitro
incubation.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
Time course of initiation of DNA
replication. Nuclei from mimosine-arrested cells were incubated
either in the absence of cytosol (open symbols), or in
cytosol from interphase cells (closed symbols) for the
indicated times. A, quantitation of the percentages of
nuclei replicating. The percentages of nuclei replicating was
determined by confocal fluorescence microscopy as detailed in the
legend to Fig. 1. B, quantitation of the amount of DNA
synthesis. The amount of replicated DNA in these reactions was
determined by [-32P]dATP incorporation and
scintillation counting.
|
|
Initiation Depends on Cyclin/Cdk Activity--
To address an
involvement of cyclin·Cdk complexes in the initiation of DNA
replication in nuclei from mimosine-arrested cells, the influence of
the specific Cdk inhibitors roscovitine and olomoucine (42, 43) was
first determined (Fig. 7). Concentrations
above 0.5 and 2 mM roscovitine and olomoucine,
respectively, inhibited replication to a background of about 10% of
the nuclei (Fig. 7, A and B). An inhibition to
50% of the maximal number of replicating nuclei was observed at about
20 µM roscovitine and 0.6 mM olomoucine (Fig.
7A), reproducing the 20-fold difference of the half-maximal inhibition by these two inhibitors of purified Cdks 1, 2, and 5 but not
of other kinases (42). In density substitution experiments, only small
amounts of DNA synthesis products of intermediate densities between LL
and HL were formed in the presence of 0.5 mM roscovitine (Fig. 7C). Together, these data strongly suggest that
initiation of DNA replication in nuclei from mimosine-arrested cells is
inhibited by roscovitine and olomoucine, and elongation of DNA
replication occurs only for relatively short distances in the 10% of
contaminating S phase nuclei present in the preparation. The identity
of the cyclin·Cdk complexes required for initiation was analyzed by
adding recombinant human cyclin·Cdk complexes to reactions in the
presence of roscovitine. Human cyclin A/Cdk2 and cyclin E/Cdk2, but not cyclin D2/Cdk6 or cyclin B1/Cdk1 could fully overcome the inhibition of
initiation by roscovitine (Fig. 7D). These results strongly suggest an essential and specific role for cyclin A/Cdk2 and/or cyclin
E/Cdk2 activity in the initiation of DNA replication in this system. As
the cytosolic initiation activity does not correlate with either cyclin
A or E protein levels (Fig. 5), I therefore investigated the
contribution of the kinase Cdk2 and the intracellular localization of
the endogenous cyclin/Cdks to the initiation of DNA replication in this
system.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Initiation depends on cyclin/Cdk2 activity
in vitro. A, inhibition of initiation
in vitro by the Cdk inhibitors roscovitine and olomoucine.
Nuclei from mimosine-arrested cells were incubated in interphase
cytosol in the presence of the indicated concentrations of roscovitine
(filled squares) and olomoucine (open squares).
Percentages of nuclei replicating were determined by confocal
fluorescence microscopy as detailed in the legend to Fig. 1.
B, representative field of replicating nuclei in the
presence of 0.5 mM roscovitine (top panel, DNA;
and bottom panel, replication). C, analysis of
reaction products performed in the absence (open squares)
and presence of 0.5 mM roscovitine (filled
squares) by density substitution as specified in Fig. 3.
D, reversion of the inhibition of initiation by recombinant
human cyclin·Cdk complexes. Nuclei from mimosine-arrested cells were
incubated in interphase cytosol in the presence of 0.5 mM
roscovitine and 0.1-0.2 µg of protein of the recombinant
cyclin·Cdk complexes as indicated. Percentages of nuclei replicating
were determined by confocal fluorescence microscopy as detailed in the
legend to Fig. 1.
|
|
Cyclin/Cdk2 Is Present in Cis and Is Not Required in Trans for
Initiation--
The contribution of the kinase Cdk2 from either
soluble extract in trans or nuclei in cis during
the initiation reaction in vitro was first analyzed by
depleting Cdks 1 and 2 from initiating interphase cytosol using
Sepharose beads coated with protein p9Cks1 (Fig.
8). Western blot analysis confirmed
depletion of Cdk1 and Cdk2 from the extract (Fig. 8A). As
control, Cdk4 was not depleted (Fig. 8A). The depleted
cytosol triggered initiation of DNA replication in nuclei from
mimosine-arrested cells as efficiently as mock-depleted cytosol (Fig.
8B), indicating that Cdk2 (and Cdk1), and hence its kinase
activity, is not supplied by the cytosol in trans in this
system. Finally, the relative localization of these cyclin/Cdks in
either cytosol or nuclei from mimosine-arrested cells was analyzed by
Western blotting (Fig. 8C). Cyclins A and E, and the kinase Cdk2 were clearly and predominantly present in salt extracts of nuclei
from mimosine-arrested cells (Fig. 8C). Protein levels of
cyclin A and Cdk2 in cytosolic and nuclear extracts from
mimosine-arrested cells were similar to the protein levels found in
cytosolic and nuclear extracts from S phase cells (data not shown).
Taken together, these data demonstrate that cyclin A/Cdk2 and cyclin
E/Cdk2 are provided by the nuclei in cis. Therefore,
initiation of DNA replication in nuclei from mimosine-arrested cells
requires nuclear cyclin/Cdk activity and an additional activity present
in cytosolic extracts from untreated cells.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Cis-requirement for cyclin A/Cdk2 and E/Cdk2
for the initiation of DNA replication in nuclei from mimosine-arrested
cells. A and B, cytosolic extract from
interphase cells was depleted with p9Cks1 beads as
specified under "Materials and Methods." A, Western blot
analysis of mock-treated (mock) and depleted cytosol ( ). Identical
protein amounts of each extract (50 µg) were separated on
polyacrylamide gels, blotted, and probed with antibodies against the
indicated proteins as specified under "Materials and Methods."
B, DNA replication in nuclei from mimosine-arrested cells
upon incubation in mock-treated and depleted cytosolic extracts. Nuclei
from mimosine-arrested HeLa cells were incubated in vitro in
the absence of cytosol (white column, buffer) and in
mock-treated and depleted cytosol (black columns, as
indicated). The percentages of nuclei replicating were determined by
confocal fluorescence microscopy as detailed in the legend to Fig. 1.
C, localization of cyclins and Cdks in mimosine-arrested
cells. Western blots of cytosolic and nuclear extracts (50 µg of
protein each) from mimosine-arrested cells using antibodies against the
indicated proteins as specified under "Materials and
Methods."
|
|
| |
DISCUSSION |
The experiments reported here demonstrated that 0.5 mM
mimosine arrests human tissue culture cells in late G1
phase at a state of competence to initiate DNA replication. Nuclei
isolated from mimosine-arrested cells serve as efficient templates for
initiation of DNA replication in soluble extracts from proliferating
human cells. The state of competence to initiate DNA replication
correlates with a nuclear localization of cyclin A, E, and Cdk2
proteins. Initiation of DNA replication in vitro depends on
nuclear cyclin/Cdk2 activity and an additional, essential and
soluble initiation activity present in the cytosolic extracts.
Replication Initiation Competent Template Nuclei--
Nuclei from
mammalian cells synchronized in the G1 phase of the cell
division cycle can initiate DNA replication upon incubation in human S
phase extracts (17, 18). The competence of isolated nuclei to initiate
DNA replication in vitro arises in late G1 phase
shortly before onset of S phase in vivo. Template nuclei were previously obtained from cells that are progressing through G1 phase after a release from a block in either mitosis
(17) or quiescence (18). As a result of the inherent degree of
asynchrony of these dynamic cell populations, only limited and varying
percentages of nuclei in a preparation initiate DNA replication in S
phase extracts. However, using release from quiescence and addition of
exogenous Cdc6 protein significantly increased the percentage of nuclei
initiating in this approach (18), but the identification of endogenous
markers for initiation competent nuclei is hampered by the relative
asynchrony of the nuclear preparation.
Here, I report on the use of template nuclei prepared from human cells
reversibly arrested in the cell cycle by 0.5 mM of the
plant amino acid mimosine. These nuclei have a G1 phase DNA content and do not contain active DNA replication forks capable of
elongating DNA replication in elongation buffer in vitro
(Ref. 34 and this paper). As control, true S phase nuclei do elongate DNA replication at existing forks under these conditions (Ref. 34 and
this paper). Nuclei from mimosine-arrested cells initiate semiconservative DNA replication reproducibly with high efficiency upon
incubation in cytosolic extracts from interphase cells (this paper).
Initiation competence of nuclei from mimosine-arrested cells correlates
with high nuclear cyclin A/Cdk2 protein levels and initiation of DNA
replication depends on nuclear, but not on cytosolic cyclin/Cdk2
activity (Fig. 8). In proliferating cells, cyclin A accumulates in the
nucleus from S phase onwards until degradation in mitosis (44). Because
of the nuclear localization of cyclin A, nuclei from mimosine-arrested
cells could be considered S phase in character. However, active DNA
replication forks clearly have not been established in these nuclei
(34), and by the stringent criterion of not synthesizing DNA, they have
to be considered pre-S phase, or late G1 phase in nature.
It can therefore be concluded that mimosine blocks proliferating cells
in a state of initiation competence before the actual establishment of
active DNA replication forks. It is also conceivable, that mimosine
arrests human cells by preventing establishment of replication forks
involving a late G1 phase checkpoint, but allowing
continued cyclin A synthesis and nuclear accumulation.
Nuclear membrane integrity is neither required for, nor inhibits
initiation of DNA replication in this system. This observation is
consistent with work on Xenopus egg extracts, where
initiation of DNA replication can be observed in the absence of a
nuclear membrane when chromatin is first incubated in cytosolic egg
extract, followed by addition of a highly concentrated nucleosolic
extract (41). In this Xenopus system, replication competent
chromatin is assembled by the cytosolic egg extract and initiation is
subsequently triggered by a high concentration of soluble nuclear
factors in the absence of an intact nuclear structure, mimicking a
nuclear environment (41). In human cell extracts, it was demonstrated that an intact nuclear membrane prevents exogenous Xenopus
Cdc6 protein from binding to chromatin and establishing premature
initiation of DNA replication in mouse nuclei (18). However, initiation of DNA replication in the absence of exogenous Cdc6 protein was not
increased or inhibited by permeabilizing the nuclear membrane at the
beginning of the incubation in a subpopulation of template nuclei (18).
The data reported here (Fig. 2) show that human nuclei from
mimosine-arrested cells do not require, and are not inhibited by the
nuclear membrane for initiation of DNA replication in human cell
extracts. Together, these data suggest that G1 phase events
of establishing the competence of nuclei to initiate DNA replication
may require the assistance of selective transport across the nuclear
membrane, however, initiation per se does not depend on it
after competence for initiation is established.
The experimental approach of using template nuclei from
mimosine-arrested cells provides an extensive and robust simplification in the experimental protocols to study human initiation of DNA replication in vitro. Preparation of competent template
nuclei involves a single synchronization step of proliferating human cells and trans-acting initiating extracts are obtained from
unsynchronized proliferating human cells. This approach will allow
future analysis of the molecular events during establishment of DNA
replication forks in a variety of human cell types. Furthermore, it
allows establishment of screening tests for novel inhibitors of the
initiation of human DNA replication. This system, however, is limited
in the analysis of earlier G1 phase events during the
establishment of competence to initiate DNA replication which lie
before the arrest point of mimosine.
Involvement of Cyclin·Cdk Complexes in the Initiation of DNA
Replication--
Initiation of DNA replication in nuclei of
mimosine-arrested cells in vitro depends on the addition of
cytosolic extract from interphase cells and requires cyclin A/Cdk2 or
E/Cdk2 activity. The evidence for Cdk dependence stems from inhibition
and rescue experiments using the specific inhibitors roscovitine and
olomoucine and is further supported by correlating initiation activity
with endogenous cyclin and Cdk proteins in the materials used.
Initiation of DNA replication in nuclei from mimosine-arrested cells
was inhibited by roscovitine and olomoucine (Fig. 7). In human
fibroblasts, both compounds arrest the cell cycle in G1
phase by inhibiting Cdk2, but not Cdk4 kinase (45). Specifically, both
compounds inhibit purified Cdk1, 2, and 5 by competing with ATP binding
at the active center of the kinases (42, 43, 46). The half-maximal
inhibition (IC50) of purified protein kinase activity
differs by a factor of about 20 between roscovitine and olomoucine
(42). This relative difference in the IC50 is also observed
for the inhibition of DNA replication in nuclei from mimosine-arrested
cells (Fig. 7), strongly arguing toward an essential requirement of
cyclin·Cdk1/2 complexes in the initiation reaction. However, the
absolute values of the IC50 differ between the two types of
assay and can be explained by the presence of excess free
ATP/Mg2+ in the crude replication reactions. Lowering free
ATP/Mg2+ in replication assays to levels used in the kinase
assays with purified proteins (42, 43, 46) did not allow DNA
replication to occur in vitro (data not shown). Therefore,
the requirement for high ATP/Mg2+ precluded determination
of the IC50 under ATP/Mg2+ concentrations of
the kinase assays.
Specificity with respect to the kinase and its cyclin partner was
analyzed by adding recombinant cyclin·Cdk complexes to in vitro replication reactions in the presence of roscovitine (Fig. 7D). These experiments demonstrated that Cdk2 complexed to
cyclin A and/or E could rescue the inhibition, supporting roles for one or both of these two kinases in initiating DNA replication in human
cell extracts (17).
However, the soluble initiation activity of cytosolic extracts from
synchronized cells did not correlate with the endogenous protein levels
of either cyclin A/Cdk2 or E/Cdk2 (Fig. 4). Cytosolic cyclin A protein
was present only in background amounts throughout G1 phase
and accumulated in S and G2 phase (Fig. 4A,
cf. Ref. 44). Cyclin E protein was absent in early
G1 but was induced maximally in mid/late G1
phase and persisted at gradually decreasing concentrations through S
and G2 phase (Fig. 4A, cf. Refs. 13 and 47). Initiation activity was greatest in cytosolic extracts throughout G1 phase (Fig. 4B), indicating that
protein levels of either or both cyclins in these extracts cannot
constitute the initiation activity of the cytosolic extracts. The
kinase Cdk2 was present in cytosolic extracts throughout the cell cycle and would therefore be available for association with the cyclin subunits to constitute protein kinase activity. However, a functional role for cytosolic cyclin A·Cdk2 and cyclin E·Cdk2 complexes in triggering initiation of DNA replication in nuclei from
mimosine-arrested cells was directly excluded by depleting Cdk2 from
interphase cytosol without loss of initiation activity (Fig. 8).
Furthermore, addition of purified recombinant human cyclin A/Cdk2 and
cyclin E/Cdk2 to replication reactions in the absence of cytosolic
extract did not initiate DNA replication in nuclei from
mimosine-arrested cells (data not shown). In any case, cyclin A/Cdk2,
and to a lesser extent cyclin E, are provided in cis by the
template nuclei from mimosine-arrested cells and could therefore
constitute the roscovitine-sensitive initiation activity in
vitro (Figs. 7 and 8). This nuclear localization of cyclins A and
E, and Cdk2 in nuclei from mimosine-arrested cells may therefore also
explain the lack of dependence on S phase-specific soluble extracts to
initiate DNA replication in this system.
In nuclei from human cells released from mitosis, cyclin A/Cdk2 and
E/Cdk2 triggered initiation synergistically (17), whereas in nuclei
from mimosine-arrested cells, both could overcome inhibition of
initiation by roscovitine independently from each other (Fig. 7D). These data may indicate that a synergistic effect of
both kinases is required for triggering initiation in nuclei from
proliferating human synchronized at earlier stages of G1
phase before the mimosine-arrest point, and both kinases may act
redundantly at later stages. However, when nuclei from mouse cells
released from quiescence were used as templates, only recombinant
cyclin E/Cdk2, but not cyclin A/Cdk2 could overcome a block of
initiation by olomoucine (18). This suggests that the requirement for
either cyclin·Cdk2 complex may actually vary with and depend on the
synchronization procedures and sources of template nuclei used.
Mitotic cyclin B1 protein was enriched in G2 phase cytosol
and was only present in background quantities in the other extracts (Fig. 4A), consistent with the intracellular localization of
cyclin B1/Cdk1 in the human cell cycle (44, 48, 49). Cyclin B1/Cdk1 protein levels in the cytosolic extracts did not correlate with the DNA
replication initiation activity of these extracts (Fig. 4) and cyclin
B1/Cdk1 does not rescue inhibition of initiation of DNA replication by
roscovitine. These data exclude a functional role for cyclin B1/Cdk1 in
triggering initiation of DNA replication in human cells, consistent
with previous data (17).
D type cyclins are expressed in response to mitogen stimulation (50)
and, consequently, cyclin D1 and its kinase partner Cdk4 were found in
all extracts of synchronized proliferating HeLa cells used here (Fig.
4A). This correlates with, and therefore does not formally
exclude an involvement of this soluble protein complex in triggering
initiation of DNA replication. A direct functional role is, however,
unlikely for the following reasons. Cyclin D/Cdk4 or 6 complexes are
inhibited a 1000-fold less specifically by roscovitine or olomoucine
than cyclin A or E·Cdk2 and cyclin B1·Cdk1 complexes (42, 43, 46).
Furthermore, recombinant human cyclin D2/Cdk6 did not rescue the
inhibition of initiation of DNA replication by roscovitine (Fig.
7D). However, these data do not exclude the possibility that
cyclin D complexes contribute indirectly to the initiation activity
present in interphase cytosol.
A Novel Soluble Initiation Activity--
Taken together, the Cdk
dependence of initiation in nuclei from mimosine-arrested cells
supports a model, where cyclin A/Cdk2 or E/Cdk2 are essential, but not
sufficient for initiation of DNA replication. They are conferred by the
template nuclei in cis. Initiation, furthermore, depends on
an additional activity present in cytosolic extracts from untreated cells.
This model is supported by the observation that cytosol from
mimosine-arrested cells lacks this soluble initiation activity (Fig.
5). However, this lack of initiation activity is not complete because
initiation still occurs in about 10-15% of the nuclei. This partial
initiation deficiency is fully overridden by addition of small amounts
of interphase cytosol from cells that are not arrested by mimosine,
restoring full initiation activity (Fig. 5). This restoration of
initiation activity can also explain the partial nature of initiation
deficiency found in cytosol from mimosine-arrested cells by postulating
the presence of residual, subsaturating initiation activity in the
extract from mimosine-arrested cells. This residual activity can derive
from the proportion of cells in the preparation, which are not at the
arrest point, but in early/mid G1 or S phase (34).
The results also suggest that the in vivo target of 0.5 mM mimosine preventing entry into S phase (34), could be
identical to the soluble initiation activity found in the interphase
cytosol from cells which are not treated with mimosine. Importantly,
this initiation activity is not dramatically regulated throughout the cell cycle, however, it accumulates through G1 phase and
peaks before onset of S phase. The identity of this activity is
currently unknown, but candidates may include one or more of activities like cyclin/Cdk activating factors, substrates for S phase-specific cyclin A/Cdk and E/Cdk kinases, or activities mediating the assembly of
replication forks from DNA replication proteins. We are currently purifying this soluble activity from cytosolic extract in order to
identify factors that link cyclin/Cdk activity to the establishment of
active DNA replication forks in human cell nuclei.
TOP
ABSTRACT
INTRODUCTION
Royal Society University Research Fellow. Tel.: 44-1223-334109;
Fax: 44-1223-334089; E-mail: tk1@mole.bio.cam.ac.uk.
