Download Francon et al, 2004

Research Article
4909
A hypophosphorylated form of RPA34 is a specific
component of pre-replication centers
Patricia Françon1, Jean-Marc Lemaître1, Christine Dreyer2, Domenico Maiorano1, Olivier Cuvier1 and
Marcel Méchali1,*
1Institute of Human Genetics, CNRS, Genome Dynamics and Development, 141, rue de la Cardonille,
2Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tûbingen, Germany
34396 Montpellier CEDEX 5, France
*Author for correspondence (e-mail: [email protected])
Accepted 14 June 2004
Journal of Cell Science 117, 4909-4920 Published by The Company of Biologists 2004
doi:10.1242/jcs.01361
Summary
Replication protein A (RPA) is a three subunit singlestranded DNA-binding protein required for DNA
replication. In Xenopus, RPA assembles in nuclear foci that
form before DNA synthesis, but their significance in the
assembly of replication initiation complexes has been
questioned. Here we show that the RPA34 regulatory
subunit is dephosphorylated at the exit of mitosis and binds
to chromatin at detergent-resistant replication foci that colocalize with the catalytic RPA70 subunit, at both the
initiation and elongation stages of DNA replication. By
contrast, the RPA34 phosphorylated form present at
mitosis is not chromatin bound. We further demonstrate
that RPA foci assemble on chromatin before initiation of
DNA replication at sites functionally defined as initiation
replication sites. Association of RPA with these sites does
Key words: Replication protein A, DNA replication foci, Xenopus,
MCM, Nascent DNA, Aphidicolin
Introduction
Replication protein A (RPA) is a stable complex of three
different subunits (p70, p34 and p11) that participates in
different cellular processes: DNA replication, recombination
and repair (Wold, 1997). The RPA70 subunit has a high affinity
for single-stranded DNA, but a DNA-binding activity that is
associated with the RPA34 and RPA11 subunits (Bochkareva
et al., 1998). Cell-cycle-regulated phosphorylation of RPA34
at the G1-S transition has been described (Din et al., 1990;
Fang and Newport, 1993; Pan et al., 1994). However, it is not
yet clear whether phosphorylation of RPA is involved in the
onset of DNA replication (Pan et al., 1995; Philipova et al.,
1996), as DNA replication efficiency is not affected by DNAdependent protein kinase (Brush et al., 1994; Lee and Kim,
1995; Pan et al., 1995) or Cdc2 (Henricksen and Wold, 1994),
two kinases involved in RPA modification.
The RPA70 large subunit alone is not sufficient for DNA
replication, and the RPA complex cannot be replaced by E. coli
single-stranded DNA-binding protein SSB (Dornreiter et al.,
1992; Walter and Newport, 2000), suggesting that the RPA34
and RPA11 subunits are necessary for the function of RPA in
DNA replication. RPA participates in the synthesis and
processing of Okazaki fragments during DNA replication in
viruses and in yeast (Mass et al., 1998; Bae et al., 2001). In
multicellular organisms, however, its participation in the preinitiation complex is currently unclear. Notably, sites of DNA
synthesis are detected as replication foci that co-localize with
RPA and the DNA polymerase δ processivity factor PCNA
(Adachi and Laemmli, 1992; Dimitrova et al., 1999), but none
of the proteins forming the pre-replication complex (e.g. ORC,
cdc6, cdt1, MCMs) exhibits such clear localization. The
significance of RPA foci is therefore debated.
In Xenopus in vitro systems, RPA is present on chromatin
before initiation of DNA replication. It first localizes at distinct
foci that might be pre-replication centers, but appears to be relocalized evenly throughout the nucleus after initiation of DNA
replication (Adachi and Laemmli, 1992). In mammalian cells,
RPA localizes to replication foci at the onset of S phase
(Brenot-Bosc et al., 1995; Murti et al., 1996), but RPA foci
were not observed in early G1 (Dimitrova et al., 1999;
Dimitrova and Gilbert, 2000). These observations have led to
the proposal that RPA foci in Xenopus may be unrelated to
initiation of DNA synthesis and may represent a nonspecific
storage of RPA bound to chromatin (Dimitrova et al., 1999).
We have investigated the association of the regulatory
subunit RPA34 with chromatin during the cell cycle and its
participation in pre-replication complexes. We show that the
regulatory RPA34 subunit is rapidly dephosphorylated upon
mitosis exit, and then associates with chromatin prior to the
initiation of DNA replication. RPA34 is detected in its
hypophosphorylated form during the whole of S phase and
assembles into detergent-resistant foci. Mitosis results in
phosphorylation of RPA34 and its loss of chromatin binding
activity. At mitosis exit, RPA foci first form on sites that are
not require nuclear membrane formation, and is sensitive
to the S-CDK inhibitor p21. We also provide evidence that
RPA34 is present at initiation complexes formed in the
absence of MCM3, but which contain MCM4. In such
conditions, replication foci can form, and short RNAprimed nascent DNAs of discrete size are synthesized.
These data show that in Xenopus, the hypophosphorylated
form of RPA34 is a component of the pre-initiation
complex.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/117/21/4909/DC1
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Journal of Cell Science 117 (21)
functionally defined as replication initiation sites. The
assembly of these foci do not require nuclear membrane
formation. We further show that RPA foci can assemble in
MCM3-depleted extracts that still contain MCM4 bound to
chromatin. Moreover, these RPA foci co-localize with stalled,
short, nascent DNAs of discrete size.
37°C. DNA was then extracted with phenol/chloroform and further
purified by gel filtration chromatography on a spin column (P6,
BioRad). DNA was then incubated with 2 units of λ-exonuclease
(Biolabs) for 4 hours at 37°C in a final volume of 40 µl. At the end
of the incubation λ-exonuclease was inactivated by heating at 70°C
for 15 minutes. Analysis of the products was done by electrophoresis
on a 2% agarose gel.
Materials and Methods
Indirect immunofluorescence assays
For analysis of chromatin-bound proteins, 10 or 15 µl samples were
diluted 10 times with 0.3% Triton X-100 in XB and incubated for 5
minutes on ice. They were fixed by addition of an equal volume of
8% formaldehyde in XB, for 1 hour at 4°C. For analysis of total
nuclear proteins, 10 µl samples were fixed with 200 µl of 4%
formaldehyde in XB. Nuclei or chromatin were centrifuged onto glass
coverslips at 1500 g for 8 minutes, through a 0.7 M sucrose cushion.
Coverslips were post-fixed for 4 minutes in cold methanol and
rehydrated for 15 minutes in PBS. After 1 hour saturation at room
temperature in PBS, 1% bovine serum albumin (BSA), coverslips
were incubated overnight at 4°C with specific antibodies. Each
coverslip was washed 5 times for 20 minutes with 0.1% Tween-20 in
PBS before incubation with secondary antibodies for 1 hour at room
temperature, followed by five 20-minute washes with PBS, 0.1%
Tween-20, and a 15 minute incubation in 10 µg/ml Hoechst 33258 in
PBS.
Antibodies
The anti RPA34-specific monoclonal antibody (324A.1) was isolated
from a monoclonal antibody library raised against Xenopus oocyte
germinal vesicle proteins (Dreyer et al., 1981) and recognizes only
dephosphorylated RPA34 (Fig. S1, see supplementary material). The
RPA polyclonal antibodies 309.112 and E-Ky were generous gifts of
Y. Adachi (Institute of Cell and Molecular Biology, Edinburgh, UK).
The 309.112 antibody recognizes both RPA70 and RPA34 (Adachi
and Laemmli, 1994) but we observed that it recognized RPA34 only
under its phosphorylated form (Fig. S1, see supplementary material).
The E-Ky antibody is specific for the Xenopus RPA70 subunit
(Adachi and Laemmli, 1992). Antibodies against Cdt1, MCM3 and
MCM4 were previously described (Coué et al., 1996; Coué et al.,
1998; Maiorano et al., 2000a). γ-H2AX polyclonal antibody was
supplied by Interchim.
Xenopus egg extracts
Interphase and mitotic (CSF) low-speed (LS) extracts were prepared
according to protocols described in detail previously (Menut et al.,
1999) and available at http://www.igh.cnrs.fr/equip/mechali/.
Replication reactions
Demembranated sperm nuclei were prepared as described (Menut et
al., 1999). Nuclei were incubated in LS extracts (1000 nuclei/µl), or
mitotic (CSF) extracts that were activated with 1 mM CaCl2. Extracts
were supplemented with energy mix (10 µg/ml creatine kinase, 10
mM creatine phosphate, 1 mM ATP, 1 mM MgCl2), 150 µg/ml
cycloheximide and reactions were incubated at room temperature, or
18°C for pulse assays. DNA synthesis was measured by [α32P]dCTP
incorporation as previously described (Menut et al., 1999). DNA
replication was also determined by immunofluorescence after 3minute pulses with 20 µM biotin-16-dUTP (Boehringer) at 18°C.
Immunodepletions were performed as described (Maiorano et al.,
2000a), except for MCM3 depletions, in which IgGs were coupled to
protein-G Sepharose beads at 4°C.
Purification and analysis of chromatin fractions
50 µl samples were diluted with 5 volumes of extract buffer (XB: 100
mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM KOH-Hepes pH 7.7,
50 mM sucrose) and pelleted by centrifugation at 5000 g for 12
minutes through a 0.7 M sucrose cushion. After removing the
supernatant, nuclear pellets were resuspended in XB, 0.3% Triton X100 and incubated for 5 minutes on ice. After a further 10,000 g
centrifugation for 2 minutes, chromatin (pellet) and nucleosolic
(supernatant) fractions were recovered.
φX-174 ss or ds DNA incubated in Xenopus egg extracts was
recovered after dilution with 5 volumes of XB, 1 mM ATP, 1 mM
MgCl2, followed by gel filtration through a Sephacryl S-400 HR
column (Pharmacia).
λ-Exonuclease treatment of nascent DNA
DNA purified from either mock-depleted or MCM3-depleted egg
extracts was incubated with 0.4 mg/ml of proteinase K for 1 hour at
Microscopy and image analysis
Confocal microscopy was performed using a Biorad 1024 CLSM
system and a Zeiss LSM 510. Images were collected sequentially to
avoid cross-contamination between fluorochromes. A series of optical
sections were collected and projected onto a single image plane in the
laser sharp 1024 software and processing system. Deconvolution
imagery was performed on cut sections with a DMR Leica microscope
coupled to a CCD Princeton Camera.
Results
Two distinct populations of the regulatory RPA34 subunit
are present at mitosis and interphase with opposite
chromatin binding activity
Xenopus egg extracts mimic most events occurring during S
phase and mitosis and are particularly suitable for biochemical
analysis of initiation of DNA replication. Xenopus metaphasearrested egg extracts were supplemented with sperm chromatin
and released into interphase by calcium addition. Fig. 1A
shows the onset of S phase, whereas RPA modifications and
binding to chromatin were analyzed during mitosis exit and S
phase in Fig. 1B. We used two different antibodies that
recognize two RPA34 populations. A polyclonal antibody
raised against the RPA complex present in mitotic extracts
(Adachi and Laemmli, 1994) (see also Materials and Methods)
recognizes both RPA70 and RPA34. This antibody recognized
RPA34 only in its hyperphosphorylated form (Fig. S1, see
supplementary material). A monoclonal antibody isolated from
a library of monoclonal antibodies raised against Xenopus
oocyte germinal vesicle proteins (Dreyer et al., 1981)
recognizes the RPA34 subunit only in its hypophosphorylated
form (Fig. S1, see supplementary material for a detailed
characterization).
Nuclei were purified by low-speed centrifugation, and
detergent-extracted to obtain nucleoplasmic (S) and chromatin
(Chr) fractions. The fractions were run onto an SDS-PAGE,
RPA is a component of Pre-RCs
4911
Fig. 1. RPA34 is present as a hypophosphorylated form during the entire S phase. (A) Demembrated sperm nuclei (1000/µl) were incubated in a
mitotic extract to which calcium was added to promote entry in S phase. DNA synthesis was followed by incorporation of [α-32P]dCTP.
(B) Nuclei were isolated and treated with 0.3% Triton X-100, to collect the chromatin-bound (Chr) and nucleosolic-unbound (S) fractions, as
described in Materials and Methods. The 309.112 polyclonal antibody (pAb) was used to reveal the RPA70 and RPA34, while dephosphorylated
RPA34 was detected with the monoclonal antibody (mAb), as described in Materials and Methods and Fig. S1 (see supplementary material).
(C) The binding of RPA to chromatin was analyzed during DNA replication and entry in mitosis induced by addition of 30 µg/ml of nondegradable B cyclin (cyclin B∆90). Nuclei were isolated and treated with 0.3% Triton X-100 to collect the chromatin-bound (Chr) and -unbound
(S) nuclear fractions, as described in Materials and Methods. In mitotic or mitotic like-extracts (+ ∆ cyclin), the nuclear envelope does not form
and the chromatin-associated (Chr) and cytoplasmic (Cyto) forms of RPA were analyzed. Fractions were analyzed by 12.5% SDS-PAGE and
immunoblotted either with the 309.112 polyclonal antibody (pAb), or the monoclonal antibody (mAb) specific for dephosphorylated RPA34.
blotted and RPA was detected using the two specific RPA
antibodies. At mitosis, RPA34 is phosphorylated and does not
bind to chromatin (Fig. 1B,C). When exit from mitosis is
induced, RPA34 is dephosphorylated before entry into S phase
(Fig. 1B, mAb, 30 minutes). Dephosphorylated RPA34 starts
to accumulate on chromatin (Chr, 30-60 minutes), where it
remains throughout S phase. The RPA70 subunit shows the
same behaviour; it does not associate with chromatin at mitosis
but does during S phase.
During a normal cell cycle, we never observed the
simultaneous presence of the two RPA34 hyperphosphorylated
and hypophosphorylated populations. Fig. 1C suggests that
hyperphosphorylation of RPA at mitosis is under the control of
cdc2-cyclin B, as addition of a non-degradable form of cyclin
B to the interphase extract was sufficient to induce a complete
disappearance of the S-phase-specific hypophosphorylated
form. Concomitantly to the hyperphosphorylation of RPA34,
the chromatin binding activity of the complex was lost.
The S-phase-specific RPA34 isoform co-localizes with
RPA70 and DNA replication sites at both the initiation
and elongation stages of DNA replication
RPA foci co-localize with replication sites during S phase,
both in Xenopus (Adachi and Laemmli, 1992; Adachi and
Laemmli, 1994) and in mammals (Dimitrova and Berezney,
2002). However, in Xenopus egg extracts, RPA foci appear to
redistribute uniformly in the nucleus during S phase (Adachi
and Laemmli, 1992; Yan and Newport, 1995). This was
suggested to be the consequence of the redistribution of RPA
along single-stranded DNA during the elongation stage of
replication. RPA foci have also been detected in ORC- and
Cdc6-depleted extracts (Coleman et al., 1996), but these
experiments were performed using membrane-depleted
Xenopus egg extracts, which are not competent to initiate
DNA replication. Contradictory results, both for the nuclear
distribution as well as for the interaction between RPA34 and
RPA70, have also been reported (Cardoso et al., 1993;
Brenot-Bosc et al., 1995; Murti et al., 1996; Treuner et al.,
1999).
We first asked whether the hypophosphorylated RPA34 form
present in S phase also assembles into nuclear foci with
RPA70. To perform this study, we used the corresponding
monoclonal antibody described above, and a polyclonal
antibody specific for RPA70 (Adachi and Laemmli, 1992). Fig.
2 shows that RPA34 and RPA70 subunits are first localized in
foci before complete nuclear membrane formation (30
minutes) and before initiation of DNA replication. A uniform
distribution of RPA was then observed during S phase (60
minutes, Fig. 2A). However, when nuclei were treated before
fixation with Triton X-100, which removes the nuclear
membrane and nucleoplasmic fraction, RPA34 and RPA70
subunits were always found in nuclear foci, most of which colocalize throughout S phase (Fig. 2B). We conclude that both
subunits RPA70 and RPA34 co-localized before initiation of
DNA replication, as well as during initiation and elongation of
DNA synthesis. Our observations on intact or detergent-treated
nuclei confirm biochemical data of Fig. 1 showing that RPA is
present as two fractions in the nucleus, one bound to chromatin
and the other unbound. The increase in nuclear staining of RPA
during S phase that we and others have observed (Adachi and
Laemmli, 1992; Yan and Newport, 1995) was due to the
unbound form that accumulates in the nucleus. By contrast, the
level of chromatin-bound RPA remains constant and present in
foci that contain both the hypophosphorylated form of the
regulatory RPA34 subunit and the catalytic RPA70 subunit,
throughout S phase.
To further confirm that RPA34 foci are active replication
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Journal of Cell Science 117 (21)
Fig. 2. Co-localization of RPA34 subunit with RPA70
and DNA replication foci. (A) Sperm nuclei were
incubated in calcium-activated egg extracts and samples
were fixed 30 and 60 minutes later and analyzed by
fluorescence microscopy. DNA was detected using Hoechst
(Aa,d). The RPA34 subunit was detected using the specific
monoclonal antibody and a Texas Red-conjugated anti-mouse
antibody (Ab,e). The RPA70 subunit was detected using the
specific polyclonal E-Ky antibody (Materials and Methods)
and a FITC-conjugated anti-rabbit antibody (Ac,f). (B) Nuclei
were treated with 0.3% Triton X-100 before formaldehyde
fixation at the indicated times, and the analysis was carried out
using confocal microscopy. RPA34 (Ba-d) and RPA70 (Be-h)
were visualized as in panel A. The overlap of RPA34 and
RPA70 signals is shown (merge). Bar, 5 µM. (C) Sperm nuclei
were incubated in egg extracts, and samples were pulse
labeled for 3 minutes with biotinylated nucleotides. Three
different nuclei are shown. RPA34 was detected using the
monoclonal antibody and a secondary FITC-conjugated antimouse antibody. Nucleotide incorporation was visualized with Texas Red-conjugated streptavidin. Analysis was performed by deconvolution
and the merge of the two signals is shown (merge, yellow). Bar, 5 µM.
sites, nuclei were pulse-labeled with biotin-dUTP and then
incubated with the RPA34-specific monoclonal antibody. Single
cut sections of nuclei, analyzed by deconvolution microscopy,
demonstrate that most biotin-dUTP foci (Fig. 2, replication foci,
in red) co-localized with RPA34 foci (Fig. 2C). We also
observed RPA34 foci (Fig. 2, green) that were not yet engaged
in DNA replication, since DNA replication within nuclei is not
perfectly synchronous. The formation of these foci is sensitive
to p21 (Cip1), an inhibitor of cdk2-cyclin E that inhibits the
initiation of DNA replication by blocking the unwinding step
(Fig. 3A,B) (Walter and Newport, 2000; Adachi and Laemmli,
1994; Jackson, 1995; Yan and Newport, 1995).
Finally, we also detected the hypophosphorylated RPA foci
when sperm chromatin is assembled in Xenopus high-speed
interphase egg extracts (HSE). These extracts cannot form
nuclear membranes and, therefore, do not permit replication
(Blow and Laskey, 1986; Mechali and Harland, 1982). The
RPA foci observed do not appear to be related to DNA repair
since γ-H2AX foci, which localize at sites of DNA damage
(Kobayashi et al., 2002; Furuta et al., 2003) (Fig. S2, see
supplementary material), were not detected (Fig. 3C). The
formation of RPA foci in HSE is consistent with the early
binding of RPA34 to chromatin (within 15 minutes), before
nuclear membrane formation and the initiation of DNA
replication in low-speed egg extracts (Fig. 1) (Adachi and
Laemmli, 1994; Coué et al., 1996).
RPA assemble at pre-replication foci
The detection of RPA in discrete chromatin foci at the transition
between formation of pre-RCs and the initiation of DNA
synthesis is in agreement with its proposed function as a singlestranded DNA-binding protein required to unwind DNA at
replication origins. However, in mammalian cells it has been
suggested that RPA foci observed before initiation of DNA
synthesis are not in nuclear structures involved in initiation of
DNA replication (Dimitrova et al., 1999). As such, it is not yet
clear whether RPA foci correspond to future replication
initiation foci, where DNA replication starts. To address this
question it is important first to identify what are the future
replication initiation foci. We therefore designed an experiment
in which we could follow the assembly of RPA on sites already
identified as replication initiation sites. We first labeled
replication initiation sites on sperm chromatin incubated in
Xenopus egg extracts in the presence of biotin dUTP and
aphidicolin. The aphidicolin block was then released to allow
S phase to proceed, leading to sperm chromatin containing
replication initiation sites functionally tagged. The scheme of
RPA is a component of Pre-RCs
4913
Fig. 3. RPA34 foci do not form in the presence of p21 but
assemble in the absence of nuclear membrane formation.
(A) Sperm nuclei were incubated in a calcium-activated
mock (u) or p21-treated (e) extract. DNA replication was
monitored by incorporation of [α32P]dCTP (Materials and
Methods). (B) The same experiment as in A except that
biotin-dUTP was used to follow DNA synthesis. Samples
were treated with 0.3% Triton X-100 before formaldehyde
fixation, and immunofluorescence analysis was carried out
with the RPA34 monoclonal antibody at the initiation (30
min) or elongation stage (60 min) of DNA replication.
(C) Sperm nuclei were incubated in low-speed egg extracts
(LSE) or high-speed egg extracts (HSE) as above. DNA
was stained with Hoechst, RPA34 was detected using the
monoclonal antibody and γ-H2AX, a marker of DNA
damage, with a specific polyclonal antibody (see Materials
and Methods; Fig. S2, see supplementary material).
the experiment is outlined in Fig. 4A. We then asked whether,
in the following cell cycle, RPA assembles at these sites that are
functionally defined as DNA replication foci, or randomly on
chromatin, before initiation of DNA synthesis. As expected,
during the first cell cycle, most RPA foci observed on chromatin
co-localized with replication foci labeled with biotin dUTP
(Fig. 4B, columns 1 and 2). At the end of S phase, as determined
by introduction of bio-dUTP during the elongation phase (Fig.
S3, see supplementary material), recombinant cyclin B∆90 was
added to induce mitosis. RPA was concomitantly released from
chromatin (Fig. 4B, column 3), while the previously labeled
replication initiation foci were still detected on condensed
chromatin. We then monitored the re-binding of RPA at 5
minute intervals, immediately after entry into a new interphase,
which was induced by calcium. Strikingly, most RPA foci that
reformed on chromatin were found to assemble on replication
initiation sites functionally tagged during the previous cell cycle
(Fig. 4B, column 4), demonstrating that RPA associates with
chromatin sites that are replication initiation foci.
To confirm these data, we used a similar protocol, except
that at the end of the first cell cycle nuclei were transferred to
a membrane-depleted egg extract (Fig. 5, high-speed extract,
HSE) in which DNA synthesis on sperm chromatin does not
occur (Mechali and Harland, 1982; Blow and Laskey, 1986)
but pre-RCs are formed (Coué et al., 1996; Walter et al., 1998).
We labeled replication initiation foci during a short period of
time in the first cell cycle to ask whether RPA assembles on all
of them during the second cell cycle. We also checked for the
absence of DNA synthesis during the nuclear transfer in the
high-speed extract (data not shown), by addition of a second
dUTP analog (digoxigenin dUTP). The results of this
experiment (Fig. 5) show that, in high-speed egg extracts, RPA
assembles on chromatin into foci that co-localize with the
replication initiation foci previously identified. Some
additional RPA foci were also detected that may represent
replication initiation sites that were not labeled during the first
cell cycle in the protocol used.
Together these experiments show that RPA foci, which form
on chromatin before initiation of DNA synthesis, are structures
functionally related to DNA synthesis.
MCM3-depleted extracts assemble MCM4 and RPA onto
chromatin, and initiate the synthesis of short nascent
DNAs
DNA replication initiation occurs through a multistep process
4914
Journal of Cell Science 117 (21)
involving the assembly of a pre-replication complex, and then
the recruitment of the MCM helicase complex (Takisawa et al.,
2000; Lei and Tye, 2001). In Xenopus, the assembly of RPA
foci was previously shown to be regulated by FFA-1, a
homologue of Werner helicase (Yan et al., 1998). However,
recent data (Chen et al., 2001) report that immunodepletion of
FFA-1 neither inhibits DNA replication nor impairs the
assembly of RPA foci. To characterize the RPA contribution to
the unwinding reaction, we have taken advantage of an antiMCM3 monoclonal antibody that completely removes MCM3associated MCM2-7 proteins from egg extracts (Maiorano et
al., 2000a). However, a discrete fraction of MCM4 remains
present even after a second round of MCM3 depletion (Fig.
6A), in agreement with the presence of different MCM
subcomplexes (Thommes et al., 1997; Coué et al., 1998;
Maiorano et al., 2000a; Mendez and Stillman, 2000;
Prokhorova and Blow, 2000). DNA replication was
blocked in the MCM3-depleted extracts (Fig. 6B), and we
confirmed the absence of MCM3 but the presence of
MCM4 bound to chromatin by immunofluorescence
analysis of nuclei (Fig. 6C).
In the MCM3-depleted extract, staining of chromatin
with the RPA34-specific monoclonal antibody shows
that hypophosphorylated RPA34 assembles into foci
(Fig. 6D). These results show that inhibition of the
assembly of the whole MCM2-7 helicase complex on
chromatin does not prevent the formation of RPA foci.
Although the presence of the whole MCM complex is
necessary for an efficient and processive helicase
activity, MCM4 appears as a crucial component of the
helicase activity (Zou and Stillman, 2000; Ishimi et al.,
2003), and is the closest homologue to the unique
MCM subunit found in Archaebacteria (Kearsey and
Labib, 1998; Kelman et al., 1999; Chong et al., 2000).
This led us to consider the possibility that pre-licensed
origins containing both MCM4 and RPA34 could
permit limited DNA synthesis at origins. As shown in
Fig. 6D, nuclei formed in MCM3-depleted extracts
show significant incorporation of the nucleotide
analog biotin-dUTP. Moreover, the biotin-dUTP
labeling was not evenly distributed in the nucleus, but
appeared as distinct foci that co-localized with RPA34
foci.
To investigate the nature of the limited DNA
synthesis observed in MCM3-depleted extracts, we
analysed the length of DNA synthesized by alkali
agarose gel electrophoresis allowing the separation of
both high molecular weight and low molecular weight
products. Synthesis due to DNA repair should lead to
Fig. 4. RPA assembles to pre-replication foci. (A) Experimental scheme.
the appearance of labeled DNA of high molecular
Sperm chromatin was incubated for 60 minutes at 22°C in a low-speed egg
weight, whereas nascent DNA synthesized at origins
extract (LSE) in the presence of aphidicolin (5 µg/ml) to slow down DNA
should lead to the accumulation of labeled DNA of
replication (95% inhibition of total nucleotide incorporation, data not shown)
short and discrete size. Fig. 7A reveals that DNA
and to label replication initiation foci with biotinylated dUTP. After a first
synthesized in MCM3-depleted extracts is mainly of
wash, fresh LSE was added and elongation allowed to proceed for 60 minutes
low molecular weight, in a distinct 150-350 bp range.
without biotin dUTP. Then purified recombinant cyclin B∆90 was added to
Importantly, this discrete population of short nascent
induce mitosis. After a second wash, calcium was added to promote the exit
from mitosis and entry in a new S phase cycle. (B) Nuclei were analyzed by
DNAs was not observed in mock-depleted extracts. It
immunofluorescence at each step of the reaction to detect DNA (DAPI
is not detected in extracts depleted of the Cdt1 protein
staining), RPA (monoclonal antibody) and dUTP incorporation. The merged
(Fig. 7B), which is absolutely required for the loading
images of RPA and dUTP are also shown. DNA damage was analyzed using
of MCM2-7 complexes at origins of DNA replication
an γ-H2AX antibody, a known marker of DNA repair (Furuta et al., 2003;
(Maiorano et al., 2000b; Nishitani et al., 2000;
Kobayashi et al., 2002) (Fig. S2, see supplementary material). In row 2,
Wohlschlegel et al., 2000).
elongation was without biotin-dUTP, but it was also followed by adding
We conclude first that in the absence of MCM3,
biotin-dUTP in a sample resulting in a homogenous dUTP staining (Fig. S3,
chromatin
containing MCM4 exhibits limited and
see supplementary material). In row 4, the reassembly of RPA was
Cdt1-dependent DNA synthesis and, secondly, that
monitored at 5 minute intervals and occurred 10 to 15 minutes after adding
nascent DNA co-localizes with RPA34 foci.
fresh LSE.
RPA is a component of Pre-RCs
Two stages in the formation of initiation complexes
containing RPA are revealed by uncoupling DNA
helicase from the DNA synthesis machinery
We further asked to what extent nascent DNA formed in MCM3depleted extracts compares with synthesis in the presence of
aphidicolin, which does not inhibit the assembly of the MCM27 helicase complex but inhibits DNA polymerase-α. The
analysis was performed at early time points following the
aphidicolin block, immediately after nuclear membrane
formation, to avoid potential artefacts induced by re-initiation
events, and samples were analyzed by urea-polyacrylamide gel
electrophoresis to increase the resolution of low molecular
weight products. The population of short DNA synthesized in
the MCM3-depleted extracts was resolved into two populations
of 120-160 nt and 200-320 nt (Fig. 8A). By contrast, in
aphidicolin-treated samples, a discrete product estimated to be
39 nt (arrow) was observed in addition to a broad population of
up to 300-500 nt DNA products. The 39 nt nascent strand
population progressively disappears during prolonged
incubations with aphidicolin, at the expense of higher molecular
weight species that slowly escape the aphidicolin block, leading
Fig. 5. RPA reassembles to pre-replication foci in high-speed egg
extracts. (A) Experimental scheme. Sperm nuclei were incubated as
described in legend to Fig. 4 except that cycle 2 was followed both in
a low-speed egg extract (LSE) and a high-speed egg extract (HSE).
(B) The assembly of RPA34 was analyzed at the entry in a new
interphase in LSE or HSE, as in legend to Fig. 4.
4915
to the formation of 100-500 nt nascent strands. The 39 nt product
resulting from the aphidicolin treatment is also degraded by
DNase (Fig. 8B) and resistant to RNase treatment although its
size is slightly decreased, suggesting a partial degradation of the
RNA primers. Such short nascent DNA products synthesized in
the presence of aphidicolin have been detected during SV40
DNA replication (Anderson and DePamphilis, 1979; Decker et
al., 1986; Nethanel and Kaufmann, 1990) but so far have not
been reported for genomic DNA replication.
Fig. 8B shows that the two nascent strand populations, at 120160 nt and 200-320 nt, observed in MCM3-depleted extracts,
were degraded by DNase I but not RNase. To further
characterize the intermediates we repeated the experiment and
assayed for the presence of RNA primers at the 5′ end of the
nascent DNAs. λ-Exonuclease, an enzyme that degrades DNA
from its 5′ end except when RNA primers are present, has been
extensively used for isolation of nascent DNA at origins of DNA
replication (Bielinsky and Gerbi, 1998; Abdurashidova et al.,
2000). Fig. 8C shows the two nascent strand populations that
accumulate only in MCM3-depleted extracts. Addition of λexonuclease results in the degradation of the upper band but not
the lower band. This result suggests that the 120-160 bp
corresponds to an RNA-primed nascent DNA. Its length is in full
agreement with the observation that the first two nascent DNA
fragments initiated at the human lamin B2 origin are close to
140 bp (Abdurashidova et al., 2000) and that the major nascent
DNA products in yeast are 125 bp (Bielinsky and Gerbi, 1999).
The upper band is likely to be the result of the first two
successive nascent DNAs linked together. RNA primers are
removed from these nascent DNA that accumulate when DNA
synthesis is arrested by the lack of the processive MCM helicase.
These data define two discrete intermediate states in the
progression of the pre-RC to the initiation complex. A
population of replication initiation intermediates of 39
nucleotides in length is synthesized by the aphidicolin-resistant
DNA polymerase-α-primase activity when DNA synthesis is
blocked by aphidicolin, whereas the DNA helicase remains
active. A second population of replication initiation
intermediates is revealed when the fully processive MCM2-7
helicase activity is inhibited. DNA synthesis starts with
synthesis of an RNA-primed DNA of 125 bp but it becomes
rapidly arrested, and is limited to the first 300 bp, probably
soon after synthesis of the next nascent DNA. The RPA34
hypophosphorylated form is present at both kinds of replication
initiation intermediates.
Discussion
Two RPA34 populations during the cell cycle with
opposite chromatin binding activity
In this paper we provide new insight into the nature and
function of the foci to which RPA localizes during DNA
replication. RPA34 cycles between two forms during cell
division. Phosphorylated RPA34 is present at mitosis as
previously observed both in Xenopus (Fang and Newport,
1993) and yeast (Din et al., 1990), but hypophosphorylated
RPA34 is the only population detected from the exit of mitosis
to the end of S phase, and it is present during both the initiation
and elongation stage of unperturbed DNA replication. The
induction of phosphorylation by a non-degradable form of
cyclin B as well as the absence of phosphorylation when
4916
Journal of Cell Science 117 (21)
Fig. 6. RPA foci assemble in MCM3-depleted extracts. (A) Western blot of Xenopus egg supernatants (S1, S2) and protein precipitates (P)
resulting from a single (S1) or double (S2) immunodepletion with either control mouse IgG (mock) or an anti-MCM3 monoclonal antibody
(Materials and Methods). (B) Sperm nuclei were incubated in mock (u) or MCM3-depleted extracts (s) for different times. DNA replication
was followed by incorporation of [α32P]dCTP. (C,D) Sperm nuclei were incubated in mock- or MCM3-depleted extracts, in the presence of
biotin dUTP, as described in Materials and Methods. Samples were treated with 0.3% Triton X-100 before formaldehyde fixation, for the
analysis of chromatin-bound proteins by immunofluorescence with antibodies specific for the indicated proteins.
synthesis of cyclin B is inhibited by cycloheximide (data not
shown) is in agreement with previous observations showing
that cdc2-cyclin B is responsible for the hyperphosphorylation
of RPA at mitosis (Fang and Newport, 1993).
The hyperphosphorylated mitotic form of RPA34 does not
bind chromatin, abruptly disappears at exit from mitosis, and
is replaced by a hypophosphorylated form. This is in complete
agreement with recent data showing that phosphorylation of
RPA34 prevents association with replication centers (Vassin
et al., 2004). We also observed inhibition of RPA34
dephosphorylation by okadaic acid (data not shown), which
suggests that phosphatase 1A/2A is involved in this
dephosphorylation. This is in agreement with the inhibition
of DNA replication observed after immunodepletion of
phosphatase 2A from Xenopus egg extracts (Lin et al., 1998).
We find that hypophosphorylated RPA34 is the only isoform
present from the exit of mitosis to the end of S phase; it binds
rapidly to chromatin and assembles in discrete nuclear foci,
before initiation of DNA synthesis and during the entire
process of DNA replication. Both dephosphorylated RPA34
and RPA70 co-localize with replication foci throughout S
phase. The homogeneous staining previously observed in S
phase nuclei was due to nucleoplasmic staining and does not
reflect the chromatin-bound RPA population.
Association of RPA with pre-replication complexes
Upon incubation of sperm nuclei in egg extracts, clear RPA foci
appear after 10-20 minutes (Adachi and Laemmli, 1992; Coué et
al., 1996), which is before formation of a nuclear membrane,
whereas DNA synthesis starts at 40 minutes, with a delay that
corresponds to the formation of the nuclear membrane and the
recruitment of cdc45-DNA polymerase-α complex onto
chromatin (Mimura and Takisawa, 1998). In S. cerevisiae, as well
as in mammalian cells, RPA does not associate with chromatin
before the onset of S phase (Tanaka and Nasmyth, 1998;
Dimitrova et al., 1999; Zou and Stillman, 2000). These
differences may simply reflect different kinetics in the formation
of replication complexes. In Xenopus egg extracts, all proteins
required for DNA replication are already present in an active form
in the extract. The RPA binding chromatin step might take place
earlier, before nuclear membrane formation, and consequently be
detected as an intermediate step, as DNA synthesis will not
proceed before the nuclear membrane is formed.
Our data also show that the RPA foci that were previously
observed in high-speed egg extracts (Adachi and Laemmli,
1992) represent replication-related foci. RPA assembles to
replication initiation foci during the S phase, disassembles from
chromatin during mitosis, and reassembles to the same foci
during the second cell cycle, before DNA synthesis is initiated.
RPA is a component of Pre-RCs
Such association suggests that the organization of
chromosomal regions for DNA replication is more
conserved than expected, even in conditions where
sequence-specific origins are not detected (Laskey and
Harland, 1982; Mechali and Kearsey, 1984; Hyrien et
al., 1995). Up to 200-300 replication initiation foci are formed
during S phase in an egg extract (Coué et al., 1996; Mills et al.,
1989), a value not so different from that of somatic cells
(reviewed by Jackson and Pombo, 1998; Dimitrova et al., 1999;
Berezney et al., 2000). As each of the foci contains several
clustered replication origins (reviewed by Berezney et al., 2000),
our results suggest that the specificity of the organization of
replication domains in replication foci does not depend on the
precise location of the origins in each replicon.
If RPA foci are pre-replication foci, why have RPA foci been
detected in extracts that have been depleted of ORCs with a
specific antibody (Coleman et al., 1996)? At present, we
envisage two main possibilities: first, ORC proteins are in large
excess in the egg extracts (Rowles et al., 1996), and a small
amount of ORC (Walter and Newport, 1997) is sufficient to
allow formation of replication complexes. A second possibility
is that ORCs are not necessary for RPA binding to DNA
replication origins because RPA does not bind to chromatin at
the same DNA element recruiting ORC. Both steps, although
apparently independent, might still be involved in the
formation of a larger initiation complex. This hypothesis is
compatible with our observation that RPA is recruited to foci
that are replication initiation foci, and recent data suggesting
that origins in multicellular eukaryotes are composite
structures involving easily unwound regions (Anglana et al.,
2003; Kong et al., 2003). Another observation in agreement
with this possibility is the inhibition of the formation of RPA
foci by p21. This cdk2-cyclin E inhibitor prevents the entry of
cdc45 and DNA polymerase-α, and consequently initiation of
DNA synthesis, but allows formation of pre-replication
complexes consisting of ORC, cdc6 and MCM (Yan and
Newport, 1995; Hua and Newport, 1998; Mimura and
Takisawa, 1998). Our favored model is that RPA foci would be
structures distinct from pre-RC, formed without requiring pre-
4917
Fig. 7. Short nascent DNAs are
synthesized in MCM3-depleted
extracts. Alkaline agarose gel
electrophoresis of DNA
synthesized in egg extracts
depleted with control mouse IgGs
(mock-depleted), anti-MCM3
(MCM3-depleted) (A) or Cdt1
(Cdt1-depleted) antibodies (B).
Depletion of the Cdt1 protein was
over 98% (data not shown).
RC proteins, which will be nevertheless used later for setting
the replication initiation centers.
It has been previously reported that RPA binding to
chromatin occurs after the Cdc45 binding step (Mimura et al.,
2000; Walter and Newport, 2000), a result that apparently
contrasts with other observations (Adachi and Laemmli, 1992;
Coleman et al., 1996) (this study). However, Mimura et al. have
shown that RPA binds to chromatin before Cdc45, but less
tightly than during initiation of DNA synthesis (Mimura et al.,
2000). They proposed that cdc45 is required for the unwinding
of DNA, which in turn leads to the tight binding of RPA to the
single-stranded DNA unwound at the replication origins. These
data, together with our results, are in agreement with two
binding modes of RPA, before and after initiation of DNA
synthesis, with the association of RPA to chromatin stabilized
at initiation of DNA replication through DNA-unwinding at the
origin. The strength of RPA binding to chromatin would be a
result of the extent of single-stranded DNA generated at the
initiation and elongation steps of DNA synthesis so that RPA
would be much more tightly associated with chromatin during
elongation than during initiation. In another report (Walter
and Newport, 2000), the binding of RPA to chromatin was
challenged with high concentrations of detergent, which
probably removed the less tightly bound RPA population in
high-speed extract. However, a nucleoplasmic egg extract that
neither depends on nuclear membrane to initiate DNA
synthesis nor forms nuclei was also used in these experiments,
which could lead to different results.
Short nascent DNAs at initiation of DNA synthesis
We showed that in MCM3-depleted extracts, in which the
processive MCM2-7 helicase complex is inhibited, limited
DNA synthesis is observed. RPA34 localizes under its
4918
Journal of Cell Science 117 (21)
Fig. 8. Discrete sizes of nascent DNAs in
the absence of MCM3 or during
inhibition of DNA replication by
aphidicolin. The DNA replication
products of MCM3-depleted extracts, or
extracts treated with 15 µg/ml
aphidicolin, were resolved by denaturing
8 M urea 6% acrylamide gel
electrophoresis. Untreated samples (–),
treated for 30 minutes at 37°C with 0.1
mg/ml RNase A (R), or with 1.3 U/ml
DNase I, are shown for both MCM3depleted or aphidicolin blocked extracts.
(B) DNA replication was followed by [α32P]dCTP incorporation. (C) The labeled
DNA replication products from mockdepleted or MCM3-depleted egg extracts
were separated by alkaline 2% agarose
gel electrophoresis, which provides less
resolution than acrylamide gel
electrophoresis but gives better separation
of the two populations of nascent DNAs.
Incubation with (+) or without (–) λexonuclease (λ-exo) is shown.
hypophosphorylated form to foci at which short nascent DNA
strands are synthesized, which is also the case during an
unperturbed S phase. Moreover, this abortive DNA synthesis
is aphidicolin-sensitive (data not shown).
The association of MCM4 with chromatin in the absence of
MCM3 has been observed both in Xenopus and human Hela
cells (Coué et al., 1998; Maiorano et al., 2000a; Mendez and
Stillman, 2000), and may represent a first stable intermediate in
the loading of the full MCM2-7 helicase complex. MCM3,
MCM5 and MCM4-7 subcomplexes have been documented
(Burkhart et al., 1995; Ishimi et al., 1996; Schulte et al., 1996;
Ishimi, 1997; Thommes et al., 1997; Coué et al., 1998; Sherman
and Forsburg, 1998; Maiorano et al., 2000a; Prokhorova and
Blow, 2000; Lee and Hurwitz, 2001), and a low helicase activity
is associated only with the MCM4-7 complex in eukaryotes
(Ishimi et al., 1996; Ishimi, 1997; You et al., 1999; Ishimi and
Komamura-Kohno, 2001; Lee and Hurwitz, 2001).
Interestingly, the MCM4 subunit seems to be the closest
homologue to the single MCM present in Archaebacteria,
which possesses the DNA helicase activity (Kearsey and Labib,
1998; Kelman et al., 1999; Shechter et al., 2000; Chong et al.,
2000). Together these data suggest that an MCM4-containing
complex can carry out some limited DNA unwinding at DNA
replication origins but that the full MCM2-7 complex is
required for processive DNA synthesis, as shown in vivo (Labib
et al., 2000). We propose a two-step assembly in which RPA
contributes to the formation of an open origin complex, while
a further processive unwinding by the full helicase complex
permits entry of additional RPA molecules and DNA synthesis
to proceed. A similar mechanism takes place in prokaryotes,
where priming of short nascent strands cannot occur without
some helicase action (Fang et al., 1999). In E. coli, the singlestrand DNA-binding protein (SSB) contributes to the opening
of OriC but not to the DNA unwinding at this early step.
We have also defined here two discrete stages of DNA
replication that are characterized by the synthesis of discrete
sizes of nascent DNAs in which chromatin-bound RPA34 is
associated. The first stage is defined when DNA replication is
blocked at the initiation stage by aphidicolin. Nascent DNAs
of 39 nt are synthesized. The second stage is revealed by the
depletion of MCM3, in which initiation of DNA replication can
proceed up to 320 nt, after which the full MCM helicase
complex is required. A discrete nascent DNA population of
120-160 nt is synthesized and contains RNA-primers, as
observed at the human lamin B2 origin (Abdurashidova et al.,
2000), whereas the second population of 200-320 nt may
represent the first two nascent DNA processed and linked, as
RPA is a component of Pre-RCs
RNA primers are removed in this process. The accumulation
of these two forms may signal abortive DNA synthesis because
of the lack of fully processive MCM helicase and possibly
specific features of chromatin at origins (Anderson and
DePamphilis, 1979; Lipford and Bell, 2001).
We are grateful to Y. Adachi for generous gift of polyclonal
antibodies and purified RPA complex. We thank M. Peter for
providing us with non-degradable B cyclin, N. Lautredou (Cellular
Imagery Regional Center) for her assistance with confocal
microscopy analysis, and P. Travo, head of the IFR4 Integrated
Imaging facility, for his constant interest and support. We also thank
D. Fisher, C. Jaulin and P. Pasero for critical reading of this
manuscript. This work has been supported by the Association pour la
Recherche sur le Cancer (ARC), the Ligue Nationale contre le Cancer,
the Foundation pour la Recherche Médicale (FRM), and Human
Frontier Science Program.
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