2114–2123 Nucleic Acids Research, 2002, Vol. 30, No. 10
© 2002 Oxford University Press
Site-specific and temporally controlled initiation of DNA
replication in a human cell-free system
Christian Keller, Olivier Hyrien1, Rolf Knippers and Torsten Krude2,*
Department of Biology, Universität Konstanz, D-78434 Konstanz, Germany, 1Genetique Moleculaire, Ecole Normale
Superieure, F-75 230 Paris Cedex 05, France and 2Department of Zoology, University of Cambridge, Downing Street,
Cambridge CB2 3EJ, UK
Received February 15, 2002; Revised and Accepted March 26, 2002
ABSTRACT
We have recently established a cell-free system from
human cells that initiates semi-conservative DNA
replication in nuclei isolated from cells which are
synchronised in late G1 phase of the cell division cycle.
We now investigate origin specificity of initiation using
this system. New DNA replication foci are established
upon incubation of late G1 phase nuclei in a cytosolic
extract from proliferating human cells. The intranuclear
sites of replication foci initiated in vitro coincide with
the sites of earliest replicating DNA sequences, where
DNA replication had been initiated in these nuclei
in vivo upon entry into S phase of the previous cell
cycle. In contrast, intranuclear sites that replicate later
in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at
the lamin B2 DNA replication origin, which is also
activated in vivo upon release of mimosine-arrested
late G1 phase cells into early S phase. In contrast, in the
later replicating ribosomal DNA locus (rDNA) we
neither detected replicating rDNA in the human in vitro
initiation system nor upon entry of intact mimosinearrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into
mid S phase. These data indicate that early origin
activity is faithfully recapitulated in the in vitro system
and that late origins are not activated under these
conditions, suggesting that early and late origins may
be subject to different mechanisms of control.
INTRODUCTION
Initiation of eukaryotic DNA replication is a tightly controlled
process. In metazoa, about 30 000 replicons are coordinately activated along the chromosomes to ensure that the entire genome
is replicated precisely once throughout S phase (1). This regulation requires the concerted action of cis-acting elements that
constitute origins of DNA replication and soluble trans-acting
factors interacting with them, eventually triggering replication
fork establishment in a spatially and temporally controlled
fashion.
In mammalian cells, only a few origins of DNA replication
have been mapped on the chromosomal DNA (reviewed in 2–4).
The origin of DNA replication that has been mapped with the
highest degree of resolution to date in human cells is located at
the 3′-end of the lamin B2 gene (5,6). The transition point from
continuous to discontinuous replication along the DNA
sequence at this origin has been determined at nucleotide
resolution (7). This origin is activated at the very beginning of
S phase (8). The DNA sequence specificity for initiation is not
so clearly defined for other human origins. For instance, in the
naturally amplified locus coding for rRNA, initiation of DNA
replication occurs at multiple sites in a much broader region
spanning the 31 kb spacer, which separates the tandemly
repeated transcription units (9–11). However, preferential sites
of initiation have been mapped by different techniques within
an ∼10 kb region upstream of the rRNA gene promoter
(9,10,12–14).
The establishment of origin specificity and timing has been
addressed by experiments involving the transfer of Chinese
hamster cell nuclei isolated at various points in the G 1 phase of
the cell cycle into replication-competent Xenopus egg extracts.
It was found that the site specificity of initiation of DNA replication in the DHFR initiation zone is established at a discrete
point in mid G1, the ‘origin decision point’ (ODP) (15). The
ODP precedes the restriction point in late G1 phase, where cell
cycle progression becomes independent of mitogen stimulation
(16). Similarly, the defining point for replication timing of the
DHFR replication origin in early S phase occurs at another
discrete point in early G1, the ‘timing decision point’ (TDP)
(17,18). The precise molecular events that constitute the ODP
and TDP are still unknown (19).
Higher eukaryotic replicons are thought to be organised as
functional clusters of five to ten synchronously activated
origins (for a review see 20). Furthermore, DNA replication is
observed to occur at discrete foci in the nucleus, as shown by
incorporation of halogenated nucleotide precursors into the
genomic DNA and detection by immunofluorescence microscopy (21,22). The patterns of replication foci are highly
dynamic during S phase. During the first half of S phase foci
are located in the transcriptionally active euchromatin,
excluding the nucleoli and nuclear periphery. This pattern is
collectively referred to as type I (22); this classification will be
used throughout this paper. In mid to late S phase foci are
located at the nuclear periphery and in perinucleolar and nucleolar
regions, referred to as type II. In late S phase replication occurs
*To whom correspondence should be addressed. Tel: +44 1223 330111; Fax: +44 1223 336676; Email: [email protected]
Nucleic Acids Research, 2002, Vol. 30, No. 10 2115
within nucleoli and satellite heterochromatic regions, referred
to as type III (22,23). Using different established cell lines, the
same progression of replication foci patterns has been
observed, but some authors subdivide the patterns into five
stages of S phase and refer to types I–V (17,24,25). Activation
of the first cohort of replication foci defines the onset of
S phase, but further activation of new foci is asynchronous and
occurs throughout the remainder of S phase (26–29). Individual
foci are active for ∼45–60 min (26,29,30) and progression from
earlier S phase to later stages depends on completion of the
earlier events (28). The spatio-temporal patterns of chromosomal replication are essentially maintained from one cell
generation to the next (17,29–31). The molecular mechanisms
underlying these controls remain to be elucidated.
We have recently established a cell-free system from human
cells that allows molecular studies of the initiation of DNA
replication in isolated nuclei (32,33). For a preparation of
active template nuclei, cells need to be synchronised in late G1
phase (32,33). This synchronisation is efficiently achieved by
a block with the plant amino acid mimosine (34,35; and
references therein), which needs to be added at concentrations
of ≥0.5 mM to proliferating human cells for successful
synchronisation in late G1 phase; lower concentrations fail to
arrest cells before onset of S phase and result in their accumulation
in S phase (35). Significantly reduced or no initiation in vitro is
observed when template nuclei are prepared from early G1 or
from G2 phase human cells, respectively (32).
In the human in vitro system, efficient initiation of semiconservative DNA replication is triggered in nuclei isolated
from mimosine-arrested cells upon addition of a cytosolic
extract from proliferating human cells (33). Essential initiation
factors have been identified in this system as G 1/S phasespecific cyclin/Cdk complexes (32,33,36). These observations
together demonstrate that cell cycle control for initiation is
maintained in this human cell-free system.
The spatial and temporal regulation of the initiation of DNA
replication in the human cell-free system has not been
addressed so far. Such an analysis is warranted by results
obtained from another model system, derived from the eggs of
the frog Xenopus laevis (37; and references therein). In the
Xenopus system, initiation is observed in an origin-specific
manner only when intact post-ODP nuclei are used as a
template (15,38). In contrast, protein-free DNA, sperm
chromatin, pre-ODP nuclei or permeabilised post-ODP nuclei
all initiate DNA replication with no requirement for specific
DNA sequences in the Xenopus egg system (15,37–39). This
in vitro behaviour mimics the sequence-independent selection
of initiation sites characteristic for the early Xenopus embryo
(40,41).
In this paper, we address the site specificity of initiation in
the human system derived from proliferating somatic cells
(32,33). We first demonstrate that the intranuclear sites of
DNA replication initiated in vitro coincide with the sites of
earliest replicating DNA sequences, where DNA replication
had been initiated in the previous cell cycle in vivo. In contrast,
chromosome domains that replicate later in S phase were not
replicated in vitro. We next demonstrate by nascent strand
analysis using quantitative real-time PCR that DNA replication
initiates in this cell-free system site-specifically at the lamin
B2 replication origin, which is also activated in vivo upon entry
into S phase. In contrast, replication intermediates within the
rDNA locus are not initiated in the cell-free system nor were
they detected when intact cells entered S phase in vivo.
However, they were detected in vivo following progression of
cells into mid S phase, suggesting that origin usage in the
human cell-free system follows the spatial and temporal
pattern as seen in S phase in vivo.
MATERIALS AND METHODS
Cell culture and synchronisation
Human HeLa-S3 and EJ30 cells were cultured as proliferating
monolayers on 145 mm plates in DMEM supplemented with
10% fetal calf serum (FCS), 100 U/ml penicillin and 0.1 mg/ml
streptomycin (Gibco BRL). EJ30 cells were arrested in
quiescence by culturing confluent plates for 7 days in DMEM
supplemented with 0.5% FCS, 2.5 µg/ml amphotericin B
(fungizone; Gibco BRL), 100 U/ml penicillin and 0.1 mg/ml
streptomycin (Gibco BRL). These quiescent cultures were
stimulated by washing and re-cultivating in DMEM containing
10% FCS.
Proliferating cells were arrested in late G1 phase by adding
0.5 mM mimosine (Sigma) from a 10 mM stock solution to the
culture medium for 24 h (35). Serum-stimulated EJ30 cells
were arrested at the G1/S border by adding 5 µg/ml aphidicolin
(Sigma) to the culture medium 8 h post-stimulation and further
cultivation for 20 h. Synchronisation was monitored by flow
cytometry of isolated nuclei (35).
Labelling of nascent DNA with halogenated nucleotides
To label DNA replicated in vivo, 20 µM 5-chloro-2′-deoxyuridine
(CldU; Sigma) was added to the culture medium of human
HeLa or EJ30 cells. Earliest replicating DNA was pulse
labelled in vivo by washing aphidicolin-arrested EJ30 cells
with phosphate-buffered saline (PBS) and releasing them into
fresh DMEM containing 20 µM CldU for 20 min, followed by
replacing the culture medium with DMEM without label. DNA
replicated in early S phase in vivo following release from a late
G1 phase block imposed by 0.5 mM mimosine (35) was
labelled by replacing the culture medium with DMEM
containing 20 µM 5-iodo-2′-deoxyuridine (IdU; Sigma) for 3 h.
DNA replication in vitro
Template nuclei were prepared by hypotonic treatment of the
cells, followed by Dounce homogenisation and centrifugation,
exactly as described previously (35). Cytosolic extract was
prepared from asynchronously proliferating HeLa cells exactly
as described (33). Protein concentrations were determined with
the Bio-Rad protein assay using bovine serum albumin as
standard.
DNA replication initiation reactions were performed as
described, with minor modifications (32,33). The reaction
mixture contained HeLa cell cytosolic extract (100 µg protein);
a buffered mix of rNTPs and dNTPs, including digoxigenin11-dUTP (dig-dUTP; Roche), an ATP-regenerating system
and 2–5 × 105 nuclei from mimosine-arrested HeLa cells. The
final reaction volume of 50 µl was adjusted with replication
elongation buffer (20 mM K–HEPES, pH 7.8, 100 mM potassium
acetate, 1 mM MgCl2, 0.1 mM dithiothreitol). DNA replication
elongation assays (35) contained the same components except
for the initiating cytosolic extract. For analysis of nascent
2116 Nucleic Acids Research, 2002, Vol. 30, No. 10
DNA strand abundance by real-time PCR and for 2D gel
electrophoresis, these in vitro reactions were scaled up 800 times
to a final volume of 4 ml. Incubation time was 3 h at 37°C.
Analysis of DNA replication products
Confocal immunofluorescence microscopy. For differential
analysis of labelled nascent DNA by confocal fluorescence
microscopy, the CldU- and IdU-specific antibody protocol
(42,43) was adapted and integrated into the labelling protocol
for DNA replicating in vitro (33,35). In detail, 2–5 × 105
isolated nuclei or an entire 50 µl replication reaction was
resuspended in 1 ml of 2% paraformaldehyde in PBS, mixed
well and fixed at room temperature for 5 min. Nuclei were
spun onto poly(lysine)-coated coverslips and washed once in
PBS and once in water. To allow immunostaining, the coverslips were incubated in 0.8 N HCl at room temperature for 30 min
and washed in water, followed by PBS and PBS containing
0.1% Triton X-100, 0.02% SDS for 2 min each. Coverslips
were blocked in blocking buffer (PBS, 2% non-fat dry milk,
0.1% Triton X-100, 0.02% SDS) for 30 min. Primary antibodies [rat anti-bromodeoxyuridine monoclonal antibody
MAS250b (Harlan Sera Lab) for the detection of CldU and
mouse anti-bromodeoxyuridine antibody 347580 (Beckton
Dickinson) for the detection of IdU] were diluted 1:10 in
blocking buffer and incubations were at 37°C for 1 h.
Secondary antibodies [Texas Red-conjugated anti-rat IgG
(ImmunoKontact) and fluorescein-conjugated anti-mouse IgG
(Amersham)] were diluted 1:100 in blocking buffer and
incubations were at 37°C for 1 h. For detection of DNA replicated
in vitro, fluorescein-conjugated anti-digoxigenin Fab fragments
(Boehringer Mannheim) were used at 1:100 dilution in
blocking buffer during the secondary incubation. For quantitative
analysis, total nuclear DNA was counterstained with 1 nM
TOTO-3 (Molecular Probes) during the secondary incubation.
Finally, coverslips were washed in PBS containing 0.1%
Triton X-100, 0.02% SDS, followed by PBS, and mounted in
85% glycerol containing 2.5% n-propylgallate (Sigma).
Confocal immunofluorescence was performed on a Leica
TCS microscope using the Leica Confocal software. For high
resolution double labelling with fluorescein and Texas Red,
excitation was with krypton and argon lasers at 488 and 568 nm,
respectively, and recording of the fluorescence signal was set
at 510–530 nm for the fluorescein channel and at 620–670 nm
for the Texas Red channel. For a quantitative analysis at lower
magnification, triple labelling with fluorescein, Texas Red and
TOTO-3 was performed with an additional excitation by a
helium/neon laser at 633 nm. The fluorescein channel was
unchanged in this case, but the Texas Red channel was adjusted
and recorded at 610–625 nm and TOTO-3 was recorded at
650–700 nm. Images were recorded as RGB files at 1024 × 1024
pixels at 8 bits/pixel. Eight averaging runs were performed per
sample. Low resolution fields were recorded through a Leica
PL APO 40× oil immersion objective and high resolution
images through a Leica PL APO 63× oil immersion objective
at 4× zoom. Images were processed via Adobe Photoshop 6.0
software using standardised brightness and contrast operations
only. All panels shown per figure were processed identically.
Real-time PCR analysis of nascent DNA strands. Nascent
DNA strands were prepared as detailed previously (44) with
minor modifications. For preparation from intact cells, ∼1 × 108
HeLa cells were trypsinised and washed twice in ice-cold 1× PBS
and once in RBS (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM
MgCl2). Cells were resuspended at ∼2.5 × 107 cells/ml in RBS
and left on ice for 5 min. Subsequently, the same volume of
RBS containing 1% NP-40 was added and incubated on ice for
a further 10 min. Then nuclei were pelleted at 2000 r.p.m. for
10 min in a swinging bucket (HB4) rotor, washed twice in RBS
and finally resuspended in RBS buffer at 5 × 107 nuclei/ml. For
preparation from isolated nuclei incubated in vitro, 0.5–1 × 108
nuclei were incubated in a scaled up in vitro assay for 2 h,
which was stopped by adding an equal volume of ice-cold
PBS. Nuclei were pelleted at 5000 r.p.m. in an SS34 rotor
for 5 min, washed once in SuNaSpBSA, pelleted again and
resuspended in RBS buffer at 5 × 107 nuclei/ml. The same
volume of 2× lysis buffer (20 mM Tris, pH 8.0, 20 mM EDTA,
2% SDS, 500 µg/ml proteinase K) was added to the preparations
and incubated overnight at 56°C. Total genomic DNA was
carefully extracted with phenol/chloroform, precipitated with
isopropanol and dissolved in TE buffer.
For denaturation, the DNA was incubated at 85°C for 10 min,
followed by chilling on ice. Denatured DNA was then loaded
onto linear neutral 5–30% sucrose gradients in TNE (10 mM
Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, pH 8.0). In a
parallel gradient, double-stranded size marker DNA (1 kb
ladder; MBI Fermentas) was loaded as a reference for nascent
DNA strand size selection. Samples were centrifuged at 20°C
in a Beckman SW28 rotor for 20 h at 26 000 r.p.m. Fractions of
1 ml were collected from top to bottom. As a reference, fractions
containing marker DNA were precipitated with ethanol, separated
on a 1% agarose gel and stained with ethidium bromide. Fractions
from the denatured genomic DNA corresponding to an average
single-stranded reference size of 1 kb were collected and
precipitated.
For quantitative analysis of nascent DNA strands in the
lamin B2 origin region the following primer sets were synthesised
and used: LB-forward (5′-ggc tgg cat gga ctt tca ttt cag-3′),
LB-reverse (5′-gtg gag gga tct ttc tta gac atc-3′), LBC1forward (5′-gtt aac agt cag gcg cat ggg cc-3′), LBC1-reverse
(5′-cca tca ggg tca cct ctg gtt cc-3′), LBC2-forward (5′-cac agc
atg cgg ctg ctg atc tg-3′) and LBC2-reverse (5′-cct ggt gcg tcc
cat ctg cct gc-3′). We chose to generate these primer sets
because other published primer sets used to amplify DNA
sequences near the lamin B2 origin (44) did not give single
PCR products using the settings for quantitative real-time
PCR. For real-time PCR, annealing temperatures were
adjusted to 66°C for the LBC1 and LB primer sets and 68°C
for the LBC2 set. Real-time PCR was performed with a Light
Cycler instrument (Roche) using a ready-to-use ‘hot start’ reaction mix (Light Cycler FastStart SYBR Green I Kit; Roche). The
mix contains Taq DNA polymerase and the fluorescent SYBR
Green I dye. PCR reactions contained 2.5 mM MgCl2 and 5 pmol
of each primer and were transferred to specialised Light Cycler
capillaries, which fit into the instrument’s adaptor. For
construction of a standard curve, DNA samples of known
concentrations were serially diluted containing 30 ng, 3 ng,
300 pg, 30 pg and 3 pg human genomic DNA. Nascent DNA
strands were added using 1/20 from the respective fractions.
Two reactions of each sample were set up in parallel for
double determinations.
Quantifications by the Light Cycler instrument were
performed at 50 PCR cycles using the settings of the standard
Nucleic Acids Research, 2002, Vol. 30, No. 10 2117
experimental protocol recommended by Roche. PCR products
were checked by melting curve analysis and gel electrophoresis. For quantitative analysis, the copy number in target
molecules was calculated by plotting the logarithm of fluorescence versus the cycle number and setting a baseline x-axis.
To produce the standard curve, the x-axis crossing point of
each standard sample was determined and plotted against the
logarithm of concentration. About 300 pg of DNA refer to 100
genomic equivalents. The amount of nascent DNA strands in
copy numbers was determined by extrapolation from the
standard curve. All calculations were carried out with the Light
Cycler software.
Neutral/neutral 2D gel electrophoresis of replication intermediates. For analysis of rDNA replication in vivo, DNA
replication intermediates from intact cells were isolated on a
nuclear matrix essentially as described (45; method E), with
minor modifications detailed below. For isolation from in vitro
reactions, 0.5–1 × 108 nuclei were incubated in a scaled up in vitro
assay for 3 h, which was stopped by adding an equal volume of
ice-cold PBS. Nuclei were pelleted at 5000 r.p.m. in an SS34
rotor for 5 min, washed once in SuNaSpBSA and stored
frozen. Thawed nuclei were washed once in cell wash buffer
(CWB; 45) containing 0.1% digitonin and further processed as
nuclei from intact cells (45; method E). Nuclear matrices
isolated from 3–5 × 107 nuclei from both sources were digested
with EcoRI and HindIIII, replication intermediates were
isolated from the digested matrices and were further purified
using one or two rounds of benzylnaphthyl-DEAE (BND)–
cellulose chromatography, as previously described (45; method E).
Additional rounds of EcoRI + HindIII digestion were
performed after each BND–cellulose column to maximise
digestion of replication intermediates. Neutral/neutral 2D gel
electrophoresis was performed as described (46), with modifications for the analysis of large fragments (47): the first dimension gel was 0.3% (w/v) agarose, run at 0.3 V/cm for 3–5 days
at room temperature; the second dimension was 0.6% (w/v)
agarose, 0.3 µg/ml ethidium bromide, run at 1.0 V/cm for 2–3 days
at room temperature. The gels were blotted in 0.4 M NaOH
onto Hybond-N+, hybridised with probes CHB and CPE or a
probe corresponding to the EcoRI fragment B of the human rDNA
locus (9) and analysed on a Fuji BAS 1000 phosphorimager.
RESULTS
De novo formation of DNA replication foci in the human
cell-free system
We first controlled for the specificity of initiation of DNA
replication in the human cell-free system (33) by introducing a
double label analysis using differently halogenated nucleotide
precursors (42; and see below). We asked whether new replication foci are formed de novo in late G1 phase nuclei that did
not replicate during the synchronisation treatment with mimosine.
The results confirmed previous observations (33) demonstrating that new replication foci were formed in vitro in ∼40%
of true G1 phase nuclei that had not synthesised DNA during
the synchronisation treatment with mimosine in vivo (data in
Supplementary Material). Results also confirmed that DNA
replication elongates in vitro in contaminating S phase nuclei
at existing forks in ∼10% of the nuclei (cf. 33), however, in
these few S phase nuclei new foci were also initiated next to
existing ones in vitro (data in Supplementary Material).
Similar percentages of nuclei initiating DNA replication foci
were obtained in control experiments where mimosinearrested cells were released from the block and allowed to
enter S phase in vivo (data in Supplementary Material).
Replication initiates in vitro at the earliest replicating
chromatin domains of the previous cell cycle
We used a double labelling protocol (Fig. 1A) to ask whether
DNA replication initiates in late G1 phase nuclei in vitro within
the chromosome domains that replicate first upon entry of
intact cells into S phase in vivo. Quiescent human EJ30 cells were
stimulated to proliferate and were subsequently arrested during
the first G1/S transition by addition of aphidicolin, an inhibitor
of DNA polymerases. Earliest replicating DNA was pulse
labelled with CldU for 20 min following release from the
aphidicolin block, and sites of incorporation were detected
with CldU-specific antibodies by confocal microscopy (42).
Further transit through S, G2 and M phase was allowed in the
absence of label and these cells were then arrested in the
following late G1 phase by addition of mimosine (35).
To control for intranuclear sites of replication initiated in the
intact cell in vivo, the mimosine-arrested cells were released
into very early S phase using fresh culture medium for 3 h (35).
Sites of DNA replication were labelled by inclusion of IdU and
detected with IdU-specific antibodies (42). We found that 68%
of the nuclei were labelled with CldU in the first S phase and
42% were labelled again with IdU upon entry into the second
S phase in vivo (Fig. 1B, right column). Thirty-two per cent of
the nuclei were not labelled at all and no nuclei were detected
with only the IdU label. The intranuclear position of the
replication foci formed in these two consecutive cell cycles
in vivo was compared by high resolution confocal microscopy using fluorescent antibodies against CldU and IdU
(Fig. 1C). Both labels co-localised at discrete sites in these nuclei
(Fig. 1C), forming a pattern of replication foci typical for early
S phase (type I; see ref. 22). These data establish that DNA
replication in vivo initiates after release from a late G1 phase block
by mimosine at the same intranuclear chromatin domains at
which S phase had initiated in the previous cell cycle.
We then used the same approach to test whether sites of
DNA replication initiated in the human cell-free system also
co-localised with sites of earliest replicating DNA in vivo.
Nuclei prepared from mimosine-arrested cells (72% were prelabelled with CldU in vivo, Fig. 1B) were used as templates for
initiation of DNA replication in vitro. Sites of DNA replication
in vitro were labelled with digoxigenin-dUTP and detected
with digoxigenin-specific antibodies (Fig. 1A). The percentage
of active S phase contaminants in this preparation was 3%, as
measured by the in vitro elongation control in buffer only
(Fig. 1B, left column). Upon addition of cytosolic extract
containing factors essential for replication initiation, an
additional 45% initiated DNA replication in vitro (Fig. 1B,
middle column; cf. 33). At high resolution, the replication
foci initiated in this reaction in vitro clearly co-localised with
sites of earliest replicating DNA of the previous cell cycle
in vivo, forming patterns of replication foci typical for early
S phase (Fig. 1D). These data indicate that the specificity for
chromatin domains where DNA replication initiates in this
human cell-free system recapitulates the spatial control
2118 Nucleic Acids Research, 2002, Vol. 30, No. 10
observed in vivo and occurs at intranuclear sites that are
conserved between subsequent cell cycles. Furthermore, the
confinement of initiation in vitro to earliest replicating DNA
sites suggests that the temporal control of S phase progression
is maintained in vitro and late replicating sites do not initiate
DNA replication prematurely in this system.
We tested this conclusion directly by pulse labelling asynchronous cells in vivo with CldU and looking for mid S phase
nuclei replicating perinuclear and perinucleolar heterochromatin,
and we then asked whether these sites overlap with sites of
DNA replication initiated in the subsequent early S phase in vivo
(Fig. 2A). Clearly, DNA replication foci were initiated after
release from mimosine in vivo at sites that are adjacent to, and
mostly exclude, perinuclear and perinucleolar heterochromatin
that had been pulse labelled in the previous mid S phase (Fig. 2B).
We then tested whether replication foci initiated in nuclei
isolated from mimosine-arrested cells upon incubation in
cytosolic extract in vitro also exclude later replicating heterochromatin (Fig. 2C). Again, replication foci initiated in vitro
are located adjacent to, and mostly exclude, later replicating
sites, forming separate patterns of incorporation (Fig. 2C).
Taken together, these data confirm that the general order of
replication initiation timing is recapitulated in the human
cell-free initiation system and chromosome domains that replicate
in mid S phase are not prematurely initiated when G1 phase
nuclei are incubated in initiating cytosolic extract.
In vitro replication in isolated human cell nuclei initiates at
specific origin sequences
To determine whether DNA replication initiates at specific
DNA sequences in this human cell-free system, we first
analysed the abundance of nascent DNA strands at an established
cellular origin of DNA replication in human cell nuclei.
Lamin B2 origin. A cellular origin of DNA replication that
initiates very early in S phase has been mapped with high
resolution to a 474 bp DNA sequence ovelapping the 3′-end of
the lamin B2 gene (LMB2) and the promoter of the PPV1 gene
on chromosome 19 in human cells (5–7,48). We used real-time
PCR to quantify the abundance of nascent DNA strands
emanating from this origin sequence, using as reference two
sites located 4 kb upstream and 3 kb downstream of the origin,
respectively (Fig. 3A). Nascent DNA strands of ∼1 kb were
first prepared from cells released from a mimosine block into
early S phase in vivo and subjected to real-time PCR using the
three primer sets outlined in Figure 3A. We found that the
lamin B2 origin sequences were enriched ∼5-fold over the
Figure 1. Intranuclear sites of initiation of DNA replication in vitro co-localise
with sites of earliest DNA replication in vivo. (A) Experimental protocol. See
text for details. (B) Quantification of initiation of DNA replication in vivo and
in vitro. As a negative control for the initiation of DNA replication in vitro,
nuclei from CldU pulse-labelled, mimosine-arrested cells were incubated in
vitro in replication elongation buffer (buffer control). The same nuclei were
incubated in initiating cytosolic extract (initiation in vitro). As a control, nuclei
released from the mimosine block in vivo were also analysed (initiation in
vivo). The percentages of total nuclei that were only pulse labelled in early S
phase with CldU (grey bars) and of those nuclei labelled with both CldU and
digoxigenin-dUTP in vitro or CldU and IdU in vivo (black bars) were scored.
Between 600 and 1000 nuclei were recorded per sample. (C and D) High resolution confocal laser immunofluorescence microscopy. Earliest replicating
DNA was pulse labelled at the G1/S boundary with CldU in vivo and sites of
incorporation were detected with anti-CldU antibodies (red images). (C) Sites
of DNA replication initiated in vivo in the subsequent cell cycle following
release from a mimosine block were labelled with IdU and detected with antiIdU antibodies (green images). (D) Sites of DNA replication initiated in vitro
in the nuclei isolated from mimosine-arrested cells were labelled with digoxigenin-dUTP and detected with anti-digoxigenin antibodies (green images).
Signals of both channels were merged and co-localisation of red and green
yields a yellow signal (merge). Representative images of nuclei are shown at
identical magnification. Scale bar 10 µm.
Nucleic Acids Research, 2002, Vol. 30, No. 10 2119
of nuclei in the preparation (cf. Fig. 1B; 33,35). Taken
together, these results show that cells arrested in late G1 by
mimosine enter S phase by activating specific origin sequences
upon removal of mimosine in vivo, thus supporting the observation that the lamin B2 origin fires very early in S phase in vivo
(Fig. 3B; cf. 5).
Then, we analysed nascent DNA prepared from nuclei of
mimosine-arrested cells incubated in cytosolic extract in vitro.
The lamin B2 origin sequences were amplified ∼10-fold over
the neighbouring sequences (Fig. 3C). These data establish that
DNA replication initiates at an early firing origin with
sequence specificity in the human cell-free system.
Figure 2. Exclusion of de novo initiated DNA replication foci from perinuclear
and perinucleolar heterochromatin replicating in mid S phase. (A) Experimental
protocol. See text for details. (B and C) High resolution confocal laser
immunofluorescence microscopy. Replicating DNA was pulse labelled with CldU
in vivo, chased into the following G 1 phase, subsequently arrested with mimosine and sites of incorporation detected by anti-CldU antibodies (red signal).
(B) Sites of DNA replication initiated in vivo following release from the
mimosine block were labelled with IdU and detected with anti-IdU antibodies
(green signal). (C) Sites of DNA replication initiated in vitro in nuclei isolated
from mimosine-arrested cells were labelled with digoxigenin-dUTP and
detected with anti-digoxigenin antibodies (green signal). Signals of both
channels were merged and representative nuclei identified by a type II pattern
of CldU incorporation are shown. Scale bar 10 µm.
neighbouring sequences 3–4 kb away from the origin (Fig. 3B,
black bars), indicating efficient activation of this origin upon
entry into S phase. This result independently confirms earlier
reports (5–7,48). As a control, we prepared DNA under identical
conditions from cells arrested in late G1 phase by mimosine. In
this preparation, the origin sequence amplified by primer set
LB was <2-fold enriched over the two neighbouring sequences
amplified by primer sets LBC1 and LBC2 (Fig. 3B, grey bars),
indicating that no efficient initiation had occurred during the
mimosine treatment in vivo. The small enrichment of the origin
sequences in this sample is most likely due to the small
percentage of S phase contaminants present in the population
rDNA repeats. We next investigated origins that fire at later
stages of S phase. The human rRNA genes are amplified ∼400
times and are clustered in tandem repeats (Fig. 4A), forming
the nucleolar organiser regions on chromosomes 13, 14, 15, 21
and 22 (9). The transcription units for the 45S precursor rRNA
are separated from each other by non-transcribed 31 kb
spacers. In human cells, DNA replication initiates at multiple
sites within the non-transcribed spacers (9). The nucleoli are
seen by confocal fluorescent microscopy to replicate in mid
and late S phase (cf. Fig. 2; 22). Replication timing of the
rDNA repeats was analysed at higher resolution taking into
account the polymorphic nature of the amplified rDNA locus
(9). A subpopulation of rDNA repeats lacking a polymorphic
EcoRI site at the promoter (Fig. 4A, bracketed symbol) has
been reported to replicate predominantly earlier than the
subpopulation possessing this polymorphic EcoRI site (14).
We investigated the generation of replication intermediates on
both variant fragments during S phase. Since the replication of
rDNA has previously been studied by 2D gel electrophoresis,
we decided to use this approach also for the present study
(Fig. 4B), rather than the nascent strand abundance assay,
which has been used successfully in investigations of the lamin
B2 origin.
Replication intermediates were prepared from late G1 phase
(mimosine-arrested), early S phase (released from mimosine
for 3 h) and mid S phase cells (released from double thymidine
block for 3 h), cut with EcoRI and HindIII and subjected to 2D
gel electrophoresis (Fig. 4C). In G1 phase, no replication intermediates were seen. In very early S phase we could only detect
a very faint simple Y arc in either the early or the late replicating variant fragments. In contrast, in mid S phase, we
detected a prominent simple Y arc on both types of fragments.
The results for the large (later replicating) variant covering the
initiation zone are shown in Figure 4C. Although we did not
detect a significant bubble arc on the late variant, a weak
bubble arc in addition to a strong simple Y arc was detected
when the blot was reprobed with probes CHB and CPE, which
detect an early replicating HindIII–EcoRI variant restriction
fragment that is centred on the region of highest frequency of
initiation upstream of the gene promoter (9; data not shown).
The coexistence of replication intermediates of both EcoRI
variant fragments in the same time point is consistent with
previous results (14). However, we have not investigated the
degree to which the two variants replicate asynchronously
under our conditions (see Discussion). Overall, these results
suggest that efficient DNA replication of the amplified rDNA
originates mainly in the non-transcribed spacer, consistent
2120 Nucleic Acids Research, 2002, Vol. 30, No. 10
with earlier results (9,14), and does not occur at the beginning
of S phase, but does so during later stages of S phase.
We therefore asked whether this replication timing in vivo is
recapitulated in the human in vitro system. Strikingly, no DNA
replication intermediates were formed in the rDNA cluster
when nuclei from mimosine-arrested G 1 phase cells were
incubated in initiating cytosol (Fig. 4D). This result demonstrates
that initiation of DNA replication in this system is restricted to
early firing origins (such as the lamin B2 origin) and that later
firing origins (such as in the non-transcribed spacer of the
rDNA) are repressed from firing in an untimely manner.
DISCUSSION
In this paper we show for the first time that a homologous cellfree system entirely derived from human somatic cells (32,33)
initiates DNA replication in isolated cell nuclei in an originspecific manner. Furthermore, replication initiation timing is
recapitulated in this system: the early firing lamin B2 origin
initiates replication upon incubation of late G1 phase nuclei in
cytosolic extract, whereas the later firing origins in the rDNA
cluster do not initiate in vitro. Therefore, the results presented
here establish the validity of the human cell-free system as a
model system to study entry into S phase in a spatially and
temporally controlled manner.
Initiation of DNA replication foci in the human cell-free
system
Our previous work has established that human late G1 phase
nuclei can be triggered to initiate semi-conservative DNA
replication upon incubation in extracts from human cells that
were synchronised in S phase (32) or, alternatively, in extracts
from asynchronously proliferating cells (33). DNA replication
is observed by confocal immunofluorescence microscopy on a
per nucleus basis. In pilot control experiments we confirmed
that late G1 phase nuclei initiate new DNA replication foci in
the absence of pre-existing active or dormant replication forks
(see Supplementary Material). New foci were established in vitro
exclusively at intranuclear sites that had replicated when cells
entered S phase of the previous cell cycle in vivo (Fig. 1).
Amongst one of those sites initiated in vitro, initiation was
specific for DNA sequences of the lamin B2 origin (Fig. 3).
This very early firing origin was identified and precisely
mapped before in intact human cells in a series of elegant
experiments (5–8) and we were able to confirm this origin
specificity in vivo for cells entering S phase upon release from
a block in late G 1 phase by mimosine (Fig. 3). From a technical
viewpoint, our observation that the origin specificity is
conserved between intact human cells and the cell-free initiation
system should allow future exploitation of the cell-free system
for the isolation and identification of earliest replicating DNA
sequences from human cells.
Our data furthermore establish that in the human cell-free
system the order of replication timing of individual replicons is
Figure 3. Site-specific initation of DNA replication at the human lamin B2 origin
in vivo and in vitro. (A) Map of the human lamin B2 origin and the primer sets
used. Three primer sets were generated for quantification of nascent DNA
strands by real-time PCR (see Materials and Methods). The primer set LB
amplifies the lamin B2 replication initiation site (cf. 5,7). The two adjacent
primer sets LBC1 and LBC2 amplify sequences 4 kb upstream and 3 kb downstream of the origin, respectively. (B) Initiation of DNA replication in vivo.
Abundance of nascent DNA strands was quantified by real-time PCR using the
LBC1, LB and LBC2 primer sets (see Materials and Methods). Nascent strands
were prepared from HeLa cells arrested with mimosine in late G1 phase (grey
bars) and from cells that were released for 3 h from a mimosine block into early
S phase in vivo (black bars). Means ± SD from two independent experiments are
shown. (C) Initiation of DNA replication in vitro. Abundance of nascent
strands from nuclei of mimosine-arrested cells incubated in a cytosolic extract
of proliferating cells was analysed as detailed for (B).
Nucleic Acids Research, 2002, Vol. 30, No. 10 2121
Figure 4. Lack of initiation of DNA replication at the human rDNA locus in early S phase in vivo and in vitro. (A) Map of the human rDNA locus (cf. 9,14). Two
of the repeated rDNA transcription units and the intergenic spacers are shown. The region coding for the 45S rRNA precursor is indicated by a white box, the 45S
rRNA transcript as a black arrow with the 18S, 5.8S and 28S rRNA coding sequences highlighted as a series of black boxes. Restriction sites for EcoRI (E) and
HindIII (H) are shown. The EcoRI site 5′ to the transcription start site is polymorphic and is indicated by brackets. The position of the hybridisation probe (EcoRI
fragment B; 9) is indicated by a black box and the position of the analysed polymorphic E/H DNA fragment is indicated by a dashed line. (B) Principle of neutral/
neutral 2D gel electrophoresis mapping of replication intermediates (46). DNA restriction fragments containing replication intermediates are first run from top to
bottom and in the second dimension from left to right. Linear fragments migrate along the lower arc and unit length fragments accumulate at a discrete spot on this
arc. Restriction fragments containing a single replication fork migrate on arc Y and fragments containg a centrally located defined origin of bidirectional replication
migrate on arc O. (C) Neutral/neutral 2D gel analysis of rDNA replication in vivo. Total genomic DNA was purified from cells in G1 phase (arrested by mimosine),
early S phase (released from mimosine for 3 h) and mid S phase (released from a double thymidine block for 3 h), enriched for replicative intermediates, cut with
EcoRI and HindIII and subjected to 2D gel electrophoresis. Replicative intermediates were visualised by hybridisation to radioactively labelled EcoRI fragment B
and autoradiography. (D) 2D gel analysis of rDNA replication in vitro. G1 phase nuclei from mimosine-arrested cells were incubated in cytosolic extract from
proliferating cells for 3 h. Replicative intermediates were isolated and visualised as detailed for (C).
not violated. We found that chromatin sites replicating in mid
S phase, such as perinuclear and perinucleolar heterochromatin,
do not initiate prematurely in this system (Fig. 2). Furthermore,
domains replicating even later, such as nucleoli, are clearly
excluded from initiating DNA replication in vitro and upon
entry into S phase in vivo (Figs 1 and 2). With regard to
sequence specificity in the nucleolar rDNA cluster, no significant
replication intermediates are formed in vitro (Fig. 4), despite
the presence of origins of replication or initiation zones in the
intergenic spacer (9–11,14). This mirrors the situation in vivo,
where these sequences are not initiated upon entry into
S phase, but replicate at later stages of S phase (Fig. 4 and data
not shown). The precise replication timing of all of the rDNA
clusters, however, has not yet been established in detail in
human cells. The origin mapping experiments were performed
on asynchronously proliferating cells (9–11). However, one
report shows differential timing of rDNA replication in
S phase: one subpopulation is replicating late and another
subpopulation is replicating apparently early in S phase (14). In
the work of Larner et al., human cells were released for 80 min
from block with a low concentration of mimosine (0.2 mM)
and replication intermediates were observed in the rDNA locus
(14). These observations are not inconsistent with ours, as 0.2 mM
mimosine arrests human cells in early S phase and not in late
G1 phase, as in the presence of 0.5 mM (35). A release from
this early S phase block for 80 min therefore leaves the cells in
mid S phase, similar to our synchronisation using release of
thymidine-arrested early S phase cells (Fig. 4C). We are therefore
tempted to argue that the earlier replicating cohort reported by
Larner et al. (14) is not replicating at the onset of S phase, but
at a later time after the G1 to S phase transition, and would
therefore be fully consistent with our observations (Fig. 4).
Determinants of initiation competence, timing and
sequence specificity
The molecular mechanisms that define and regulate the timing
of origin firing during S phase are only beginning to be understood in metazoa (19). At discrete stages during G 1 phase
progression, replication timing and origin sequence specificity
are established. The replication timing of chromosome
domains is established in the nucleus at the TDP (17,18),
which takes place 1–2 h post-metaphase in early G1, when
chromosome domains reposition within the interphase nucleus
following exit from mitosis (17). In the nuclei of mimosinearrested human cells used in our study, proper replication
timing is maintained in vitro and in vivo (Figs 1–4). These
observations suggest that mimosine arrests human cells after
execution of the TDP. Usage of specific DNA sites as replication origins is established at the ODP in the nucleus at 3–4 h
after metaphase in mid G1 phase (15). Nuclei from mimosinearrested cells used as templates in the human system in vitro
can initiate DNA replication at a specific origin DNA sequence
which is also used in vivo upon entry into S phase (Fig. 3),
suggesting that mimosine arrests human cells after execution
of the ODP. The experiments identifying the ODP and TDP
made use of isolated hamster cell nuclei incubated in initiating
extract from Xenopus eggs (15,17,18). Subsequent work
confirmed that the ODP also operates in intact hamster cells
2122 Nucleic Acids Research, 2002, Vol. 30, No. 10
in vivo (49). Therefore, constraints defining replication timing
and origin specificity were associated with the hamster nuclei
in cis and soluble initiation factors were supplied by the
Xenopus extract. By analogy, we conclude that constraints
specifying timing and site specificity of initiation in the homologous human system used in our studies are also associated
with the late G1 phase template nuclei in cis and soluble initiation factors are supplied by the human cell extract.
A variant model of the differential timing of replication
origin activation in our in vitro system could be built, postulating
that mimosine blocks cells part way through a multistep initiation
cascade, where early firing origins would be more advanced
than later firing origins. Cytosolic initiation extract, in this
model, would only support later steps of this cascade, with the
result that early origins fire and late origins do not fire. We
cannot rule out this scenario at present, and ongoing work in
the laboratory of one of us (T. Krude) is aimed at dissecting
the molecular steps of initiation in vitro by fractionation and
reconstitution experiments of the initiating cell extract.
In any case, the competence of isolated human cell nuclei to
initiate DNA replication in vitro is established in vivo during
later stages of G1 phase progression. Nuclei prepared from
early G1 phase human cells (32) or from rodent cells released
from quiescence into G1 phase for <16 h (50) are relatively
poor templates for initiation in human cell extracts. These nuclei
lack factors that confer replication initiation competence (and
hence preclude analysis of origin specificity and timing). In
contrast, efficient initiation is observed in nuclei prepared
either from dynamic populations of cells progressing through a
narrow window of competence in late G1 phase, just before the
onset of S phase in vivo (32,36,50), or from human cells
arrested in late G1 phase with mimosine (33,35,36). These
observations indicate that the initiating human cell extracts
require a late G1 phase-specific structure within template
nuclei to trigger initiation and cannot, unlike Xenopus egg
extracts, induce replication on any DNA sequence or
chromatin structure.
Taken together, we conclude for the human cell-free system
used here that the constraints which dictate replication
competence, replication timing and origin sequence specificity
have all been assembled in vivo, before preparation of template
nuclei, and are associated with the nuclei in cis.
Implications from a yeast cell-free initiation system and
outlook
In the budding yeast Saccharomyces cerevisiae, DNA replication initiates in the chromosomal context at genetically
defined short specific origin DNA sequences known as autonomous replicating sequences (2). The molecular mechanisms
ensuring a controlled once per cell cycle firing of these origins
have been characterised in detail (51). In a cell-free system
derived from yeast cells, origin-specific initiation was
observed in isolated G1 phase nuclei upon incubation in
S phase nuclear extract (52). Initiation depended furthermore
on the prior assembly and presence of the pre-replication
complex including the origin recognition complex (ORC) and
the protein kinase Cdc7-Dbf4 in the template nuclei (52,53).
Amongst the soluble initiation factors required from the
nuclear extract was the activity of yeast cyclin Clb/Cdc28
complexes (53).
In the human system, a strict requirement of S phasepromoting cyclin/Cdk activity for initiation has already been
established (32,33,36), and ongoing research is focused on
identifying and purifying additional initiation factors from the
cytosolic extract. Our demonstration of origin specificity in the
human system (this paper) adds another apparent layer of similarity of these two systems, despite the different organisation
and sequence requirements for origin activity between yeast
and man (2,3). The reason for this similarity is most likely
associated with the requirement for an initiation-competent
nuclear structure assembled in vivo as templates for initiation
in cell extracts.
In human cells, the homologues of the yeast pre-replication
complexes including the ORC, Cdc6 and MCM proteins are
assembled following exit from mitosis and are bound to
chromatin in late G1 phase (51). Preliminary western blot
experiments confirm that this is also the case for the mimosinearrested, late G1 phase cell nuclei used as templates in the
human system (D. Szüts and L. Kitching, personal communication). Cross-linking and quantitative chromatin immunoprecipitation analysis demonstrated that the ORC is bound to
the initiation site of the human lamin B2 origin (54). In addition,
we recently identified two novel binding sites for the ORC in
human chromatin that coincide with DNA sequences which
function as origins of DNA replication, suggesting that the
ORC may mark initiation sites for genome replication in
general (54; C.Keller, E.Ladenburger, M.Kremer and R.Knippers,
submitted for publication). Future experiments, which have
now become possible in vitro through the cell-free system,
should be aimed at studying in molecular detail the association of
soluble initiation factors with pre-replication complexes bound
to initiation-competent chromatin at human origin sequences.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We thank Cristina Pelizon and Dávid Szüts for critically
reading the manuscript and Ron Laskey and Tony Mills for
discussions. We also thank Jim Sylvester (DuPont Hospital for
Children, Wilmington, USA) for the generous gift of rDNA
probes. This work was supported by Cancer Research UK
(grant SP2546/0101 to T.K.), the Bundesministerium für
Bildung und Forschung, BMBF (Förderkennzeichen 01 KW
9708 to R.K.) and the Association pour la Recherche sur le
Cancer and the Ligue Nationale contre le Cancer, Comité de
Paris (to O.H.).
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