Replicon Clusters: Labeling Strategies
for the Analysis of Chromosome
Architecture and Chromatin Dynamics
In all eukaryotes, the maintenance of genetic
integrity requires that DNA replication is controlled
precisely to ensure that a perfect set of chromosomes
passes to each of the daughter cells during cell division.
Mammalian chromosomes are so long that DNA
synthesis must initiate at many positions on each in
order to complete S phase in the observed time
framemtypically 8-10 h. Direct observation of sites of
DNA synthesis on spread DNA fibres shows that initiation
events are spaced roughly 150 kb apart, giving
~1000 on a typical human chromosome. However, the
initiation events are not performed simultaneously at
all sites at the onset of S phase. Instead, small groups
of origins within replicon clusters are activated at the
onset of the replication programme such that only
about 10% of possible origins operate at this time.
Replication proceeds from these early origins until
some unknown switchmprobably related to changes
in chromatin structure that arise during this first phase
of synthesismallows new sets of replicons to be activated.
Different banks of replicons are activated in this
progressive fashion until the replication process is
During each phase of synthesis small clusters of
replicons are activated together (Fig. 1). These clusters
typically contain two to five adjacent replicons and
appear to form stable units of chromosome structure
with ~0.5-1 Mbp DNA (Jackson and Pombo, 1998; Ma et al.
, 1998; Zink et al.
, 1998; Cremer and Cremer, 2001).
The ability to label replicon clusters in mammalian
cells to reveal DNA or replication foci provides an outstanding
opportunity to analyse many aspects of chromosome
architecture and chromatin function. The
basic principle behind the approach is facile and relies
on the use of modified DNA precursor analogues that
can be incorporated into DNA in place of the natural
precursor and subsequently detected to reveal sites
(BrdU) was the
first compound to find common use in this approach
(Dolbeare, 1995). BrdU can be added to culture
mediummor indeed introduced by injection into
whole animalsmand is incorporated into DNA in place
of thymidine. Patterns of incorporation can subsequently
be visualised. Initially, low resolution techniques
were used that relied on the variable
biochemical properties of the BrdU-containing chromatin.
The subsequent use of immunostaining techniques
using antibromo antibodies (Gratzner, 1982)
and fluorescence-based detection systems revolutionised
the analysis of replicon clusters labelled in this
way. In the seminal experiment, Nakamura et al.
used BrdU incorporation and indirect immunofluorescence
to show that early replication in rat embryonic
fibroblasts occurred at some 130 sites that were
each estimated to contain at least 10 active replicons.
Further studies have defined a replication programme
(Fig. 2), with characteristic patterns of synthesis correlating
with the duplication of specific regions of the
genome at different times during S phase (Nakayasu
and Berezney, 1989; Humbert and Usson, 1992;
O'Keefe et al.
, 1992; Hozák et al.
, 1994). The activation
of specific groups of replicons also correlates with the
structured assembly of active replication centres-also called replication "factories" (Hozák et al.
the vicinity of active foci. These replication factories
are assembled transiently at the appropriate sites in
response to factors that determine the progress of S
phase. The distribution of active sites appears to be
influenced by fundamental features of nuclear structure.
Many proteins involved in the replication
machinery have targeting signals that direct their association
with the active sites. The sites are dynamic,
however, and can be shown using green fluorescent
protein (GFP)-tagged replication proteins, such as
proliferating cell nuclear antigen (PCNA) (Leonhardt et al.
, 2000), to assemble shortly before synthesis begins
and disassemble as replication within a particular
factory is complete.
|FIGURE 1 Chromosome territories in mammalian cells. HeLa
cells were labelled with BrdU at the onset of S phase for 20min
(A and B), 3 h (C), and 10 h (D). Cells were attached to glass slides
using the standard chromosome spreading technique, and BrdUcontaining
sites were visualised by immunofluorescence immediately
after labelling (A) or 5 days later (B-D). Note the structure of
individual replication foci and their organisation in individual chromosome
territories (B-D). (D) Individual territories are labelled
throughout S phase so that the boundary of each territory is seen.
Bar: 2.5µm. For details, see Jackson and Pombo (1998).
Chromosome Structure and Dynamics
|FIGURE 2 The S-phase programme. During S phase, different
classes of DNA elements are replicated at specific times. Chromatin
with the majority of transcribed genes is replicated over the first
~4h of the S phase. During this period, active sites of DNA synthesis
are in discrete foci dispersed throughout the nuclear interior
(A-C). These are designated early or type 1 replication patterns.
During mid-S phase (D-F) replication switches to peripheral inactive
chromatin. These mid-S phase patterns are also referred to as
type 2 (D and E) and type 3 (F) patterns. During late S phase (G-I)
the replication of extended blocks of constitutive heterochromatin
occurs throughout the nuclear interior. These late S phase patterns
are also referred to as type 4 (G-H) and type 5 (I) patterns. Encapsulated
HeLa cells were synchronised at the onset of S phase, and
replication sites were labelled at 1-h intervals for 10 h. Images shown
are replication sites labelled in permeabilised cells using biotindUTP.
For details, see Hozak et al. (1994) and O'Keefe et al. (1992).
Bar: 5 µm.
Mammalian chromosomes are highly structured.
This is evident from the fact that their condensation
during mitosis occurs in such a way that specific chromosomes
from different cell types can be readily recognised if they are stained in specific ways. The
banded chromosomal patterns revealed by stains such
as Giemsa correlate with differences in gene density
(transcriptionally active R bands have roughly fourfold
the gene density of G bands), slight differences in
base composition (R bands are commonly 3-5% more
GC rich), and variations in chromatin structure (antibodies
that recognize histone modification associated
with gene activity stain R bands). Fundamental features
of this organization persist during interphase
when individual chromosomes maintain spatially discrete
nuclear "territories" (Cremer and Cremer, 2001).
In mammalian nuclei, chromatin is locally dynamic
but only over short distances, typically <100 nm. Longrange
movement is constrained, and the best models
for the corresponding organisation predict randomly
arrayed structural subunits of roughly 1Mbp DNA (Cremer and Cremer, 2001), the typical size of DNA
foci. The possibility that clusters of replicons act as
stable cohorts that determine chromosome architecture
and drive critical aspects of chromatin function,
such as the replication programme, is an exciting
insight that emphasises the importance of structurefunction
In order to study this in living cells, strategies have
been developed to label DNA foci with fluorescent precursor
analogues so that it is now possible to perform
detailed studies of the dynamic relationships between
different foci and explore if these structures dictate
fundamental features of chromosome structure and
II. MATERIALS AND
Solutions are prepared in molecular biology grade
water from BDH Laboratory SupplieS (Cat. No.
443847D). Cell culture media from Sigma-Aldrich are
modified as required by the cells under study. For
many purposes it is convenient to use HeLa cells
grown in Dulbecco's MEM (Cat. No. D 5546) supplemented
with penicillin and streptomycin (Cat. No. P
0781), sodium pyruvate (Cat. No. S 8636), glutamine
(Cat. No. G 7513), and 5% foetal bovine serum (Cat.
No. F 7524). Reagents for in vitro
labelling are from
Sigma-Aldrich: potassium acetate (Cat. No. P 5708),
potassium chloride (Cat. No. P 9333), disodium hydrogen
phosphate (Cat. No. S 7907), potassium hydrogen
phosphate (Cat. No. P 9791), magnesium chloride (Cat.
No. M 1028), adenosine triphosphate (Cat. No. A 7699),
dithiotheritol (Cat. No. D 5545), phenylmethylulfonyl
fluoride (PMSF; Cat. No. P 7626), deoxynecleoside
triphosphate set (Cat. No. DNTP-100), nucleoside
triphosphates (Cat. Nos. A 6559; C 8552; G 3776;
U 1006), phosphate-buffered saline (PBS; Cat. No. P
4417), Triton X-100 (Cat. No. T 9284), saponin (Cat. No.
S 7900), digitonin (Cat. No. D 1407), lysolecithin (Cat.
No. L 4129), DNase I (Cat. No. D 7291), hydrocholric
acid (Cat. No. H 1758), sodium borate (Cat. No. B
0127), acetic acid (Cat. No. A 0808), methanol (Cat. No.
M 3641), low melting agarose (type VII-A; Cat. No. A
-lysine (Cat. No. P 4707), bovine serum
albumin (fraction V; Cat. No. A 4503), Tween 20 (Cat.
No. P 7949), trypan blue (Cat. No T 8154), Hanks' balanced
salt solution (HBSS; Cat. No. H 9269), and 4',6-
diamidine-2-phenylindole dihydrochloride (DAPI;
Cat. No. D 8417). Inhibitors used for cell cycle studies
are from Sigma-Aldrich: thymidine (Cat. No. T 1895),
aphidicolin (Cat. No. A 0781), nocodazole (Cat. No. M
1404), and colcemid (Cat. No. D 7385). The protease
inhibitor set (Cat. No. 1 206 893) and FuGene 6 (Cat.
No. 1 815 091) are from Roche Applied Science. Liquid
paraffin (Cat. No. 29436) is from BDH. The electron
microscopy grade 16% paraformaldehyde solution is
from Electron Microscopy Sciences (Cat. 15710-S). Vectashield
is from Vector Laboratories (Cat. H 1000).
TOTO III iodide is from Molecular Probes (Cat. T-
3604). Different DNA precursor analogues that may be
used to label DNA foci are indicated in Table I, and
antibodies used for indirect immunolabelling are
detailed in Table II. Plastics for cell culture are from
Nunc, and prewashed glass slides and coverslips are
from BDH. Culture dishes for live cell imaging are
from IWAKI (Cat. 3911-035) or MatTek Corporation
(Cat. P35GC-0-14-C), and coverslips with location grids
are from MatTek Corporation (Cat. P35G-1.5-7-Cgrid).
Radioactive nucleotides are from Amersham
The routine procedures described later are commonly
used to analyse replication foci
at the single cell
level. Accordingly the output will be recorded using
light microscopy with epifluorescence capabilities.
Many commercially available light microscopes are
capable of generating the necessary output; the particular
choice will depend both on the instrumentation
that is readily available and on the specific aims of
the analysis. For high-resolution work, an advanced
machine such as the Zeiss LSM 510 or equivalent from
other manufacturers should be used. For many applications
it will be adequate to use a much simpler
microscope equipped with a CCD camera. Certain
labelling strategies can be adapted to study cell cycle
parameters using flow cytometry techniques. Finally,
if specific emphasis needs to be placed on the extent of
DNA synthesis, it is often necessary to perform a single
cell analysis in conjunction with radiolabelling. The
incorporation of (32
p)dNTPs into DNA is measured
using a scintillation counter.
A. Labeling Replication Foci in Cells
Growing in Culture
5-Bromo-2'-deoxyuridine is phosphorylated by cells
to give BrdUTP and this precursor is incorporated into
DNA in place of dTTP.
- Prepare appropriate culture medium-as
required by cells under investigation.
- Prepare stock solution of desired replication precursor
analogue (Table I; e.g., BrdU) in medium. For
most purposes, it is convenient to prepare a 100× stock.
Labelling is usually performed at 10-50µM BrdU,
depending on the duration of labelling. Higher
concentrations may be used for very short pulses,
although concentrations in excess of 50µM may
lengthen the cell cycle. IdU is much less soluble than
BrdU and should be made as a 10× stock.
1. Adherent Cells
2. Nonadherent Cells
- Place clean, sterile 13-mm glass coverslips in a
60-mm petri dish and coat with poly-L-lysine by
applying a drop of sterile 0.01% solution for 1 h.
- Rinse coverslips with medium and seed ~2 × 105 cells in 2ml fresh medium and grow for 16-24h or
until cells reach ~50% confluency.
- Replace medium with fresh medium containing
10-50µM BrdU and incubate for 1-10h. The concentration
should be modified according to the labelling
time requiredmuse lower concentrations for longer
labelling periods. For very short pulse labels, concentrations
in the range of 50-200µM BrdU can be used.
- Rinse samples in PBS at room temperature and
prepare for immunolabelling.
Nonadherent cells can be labelled directly in suspension
in medium. However, for many in vivo
vitro applications it is convenient to use cells encapsulated
in agarose microbeads
(Jackson and Cook, 1985).
For some applications, particularly high-resolution
analyses using electron microscopy (e.g., Hozak et al.
1993), it is also convenient to perform experiments on
adherent cells that are encapsulated after removing
from the surface on which they normally grow. Use the
following steps to encapsulate cells.
- Warm 10 ml liquid paraffin to 37°C.
- Heat 0.25g low-gelling agarose in 10ml PBS at
~95°C until dissolved and cool to 37°C.
- Resuspend ~107 cells (a final cell density of 2 × 106 cells/ml beads is ideal for this application,
although a wide range can be used) in 4ml PBS at
37°C in a 100-ml round-bottomed flask.
- Add 1 ml agarose solution at 37°C to 4 ml cell suspension
at 37°C and mix thoroughly.
- Add 10ml paraffin at 37°C, seal flask with plastic
film, and immediately shake (by hand or at
800 cycles/min using a flask shaker) until a creamy
emulsion forms (~15s).
- Cool flask by periodic rotation in ice-cold water for
10min; this allows spherical droplets of molten
agarose suspended in the paraffin to gel.
- Add 35 ml ice-cold PBS, mix, and transfer to a 50-
ml plastic centrifuge tube.
- Pellet microbeads by spinning at 1000rpm on a
bench centrifuge at 20°C.
- Aspirate the supernatant and wash pelleted
microbeads once in PBS. If some beads remain at
the water/paraffin interface, remove most paraffin,
mix thoroughly, and respin.
- Encapsulated cells can now be regrown in medium
or permeabilised directly.
Incorporate BrdU or alternative precursor analogue
into encapsulated cells as follows.
3. Labeling in vitro
- Use cells adapted for suspension culture or cells
encapsulated in agarose microbeads at a density of ~2 × 106/ml and grow in fresh medium for at least 1 h.
- Add 10-50µM BrdU for 1-10h; use a higher
concentration if shorter pulse times are required (see
- As an optional step, double labelling can be
performed using iododeoxyuridine (IdU) and
chlorodeoxyuridine (Aten et al., 1992) or IdU and BrdU
(Jackson and Pombo, 1998). After the first pulse, e.g.,
20µM BrdU for 1 h, wash samples once in fresh
medium by centrifugation (1000 rpm) and resuspension
and add medium with a second precursor for a
second pulse, e.g., 20 µM IdU for 1 h.
- Wash in PBS at room temperature and prepare
for immunolabelling. The routine procedure for
washing agarose microbeads is facile. Beads pellet
readily during centrifugation at ~1000 rpm in a benchtop
centrifuge for 1-2 min. The supernatant can then be
aspirated (with care no beads should be lost) and the
bead pellet resuspended in any solution of choice.
For many applications, labelling in vitro
provides an appealing versatility. A
major advantage of this option is that replication elongation
rates can be manipulated precisely by adjusting
the concentration of the precursor pools, incubation
time, and temperature. It is also convenient to use a
much wider variety of modified precursors (Table I),
as permeabilised cells do not restrict access of the
dNTPs to the nucleus. The chromatin structure is especially
sensitive to changes in its ionic environment, and
the choice of the buffer to use with permeabilised cells is particularly important. Many buffers are in common
use, but the following "physiological buffer" (PB) is
useful for preserving critical features of nuclear structure
Comments and Pitfalls
- Prepare PB containing 100 mM KCH3COOH,
30 mM KCl, 10 mM Na2PO4, 1 mM MgCl2, 1 mM Na2ATP, 1 mM dithiothreitol, and 1 mM PMSF and add
100 mM KH2PO4 as required to give pH 7.4. Protease
and nuclease inhibitors should be added to suit the
demands of particular experiments.
- In PB, prepare 10× concentrated initiation mix to
give the following final concentrations: 250 µM dATP,
250 µM dCTP, 250 µM dGTP, 100 µM CTP, 100 µM GTP,
100 µM UTP, and 10-100 µM TTP analogue (Table I),
plus MgCl2 at a molarity equal to that of the
Various combinations of monovalent anion can be
used. Different chloride/acetate/glutamate (or polyglutamate)
combinations support similar rates of replication.
However, acetate/glutamate is preferred to the
, which is more damaging to the tertiary
protein structure. The concentration of divalent cations
must be controlled carefully. As little as 0.5 mM
causes the visible (by EM) collapse or aggregation
of chromatin. The equimolar Mg/ATP combination
used here preserves the chromatin structure
and supports the action of Mg-dependent enzymes.
Dithiothreitol, protease inhibitors, and ribonuclease
inhibitors protect the sample and preserve cell
Comments and Pitfalls
- Prepare cells on coverslips or encapsulated in
agarose microbeads as described earlier.
- Wash samples in ice-cold PB to remove medium
and permeabilise for ~2min using 0.01-0.1% of a
- Wash cells three times in ice-cold PB to remove the
- Incubate coverslips or microbeads in PB at 33°C for
- Add one-tenth volume of 10× IM, mix, and incubate
at 33°C for 2-60 min.
- Wash three times in >10 volumes ice-cold PB and
proceed to fix and label.
The choice of detergent will depend on the requirements
of a particular experiment. We commonly use saponin at 0.01%, as this preserves nuclear structure
and supports endogenous levels of DNA or RNA synthesis.
The extent of lysis is critical. To define conditions,
use a twofold dilution series of detergent in PB
and assess the level of permeabilisation using trypan
blue exclusion (add 50 µl 1% trypan blue in PB to a coverslip
or 50 µl packed microbeads; after 2min, inspect
by light microscopy; score percentage permeabilised,
dark-blue cells). Choose the detergent concentration
that permeabilises ~95% cells. If cells detach from coverslips
during washing, use a lower concentration
of detergent. The following (and related) detergents
can be used (guideline concentrations are indicated):
0.02-0.05% Triton X-100; 0.01-0.02% saponin;
0.01-0.02% digitonin; and 0.02-0.05% lysolecithin.
The concentration of modified precursor and duration
of labelling can be adjusted to suit individual
requirements. The following provides some guidelines.
Fifteen-minute incubations with 20 µM
digoxigenin-coupled precursors give good indirect
immunofluorescence signals, and longer incubations
give correspondingly stronger signals. Five- and 2-min
incubations with 100 µM
biotin-16-dUTP allow detection
by light and electron microscopy, respectively,
using standard detection protocols. Incorporated
labels can be detected after ~30-min incubations
with 20 µM
fluorescent precursors. Overall levels of
incorporation can be quantitated by incorporating a
radioactive tracemtypically [Pg2]dCTPminto the reaction;
use ~50µCi/ml [P32
]dCTP and reduce dCTP pool
to ~10 µM
If inhibitors are to be used, incubate them for 15 min
at 0°C prior to the addition of 10× IM.
B. Chromosome and Nuclear Spreads
Samples labelled in suspension can be fixed onto a
glass surface using a standard chromosome spreading
technique (Fig. 1).
- Hanks balanced salt solution (HBSS).
- Prepare 0.075M KCL in distilled water.
- Prepare 3:1 (v/v) methanol:acetic acid fixative.
Comments and Pitfalls
- In a 10-ml plastic tube, wash cells labelled with
DNA precursor analogue once in HBSS at room temperature.
Resuspend the cell pellet in 0.075M KCl
(0.5-1 × 106 cells/ml) and incubate at 37°C for 15min
to swell the cells.
- Using 2-5ml of swollen cell suspension in a
10-ml plastic centrifuge tube, prefix cells by adding
3 drops/ml of methanol/acetic acid (3:1)-add the fixative dropwise from a Pasteur pipette while agitating
the cell suspension gently. Leave the mix at room
temperature for 15 min.
- Pellet cells at 1000rpm, aspirate the supernatant
gently, and resuspend the pellet (by gentle agitation of
the tube) in methanol/acetic acid (3:1). Stand at room
temperature for 1 h.
- Pellet the sample once more and resuspend cells
in fresh methanol/acetic acid at 2 × 106 cells/ml.
- Glass slides should be rinsed in detergent (e.g.,
Decon 90), washed extensively in double-distilled
water, and chilled to 4°C in water. Take a chilled slide,
drain off excess water, and hold the slide at a ~30° angle against a work surface. Immediately drop 2-3
drops of the cell suspension from a Pasteur pipette
onto the centre of the slide. The organic solvent and
fixed cells will spread in the water film to cover most
of the slide.
- Place the slide on paper towels soaked in iced
water and allow to dry. The dried slides can be stored
in a fridge for many days before immunolabelling.
If the analysis requires significant numbers of
mitotic cells, it is usual to begin by growing cells for
30-60min in medium supplemented with colcemid
(50ng/ml). This drug disrupts microtubule function
and blocks cells in metaphase.
Hydrodynamic properties of the drying spread
have a significant impact on the quality of mitotic
spreads. Reproducibly high-quality spreads are
usually obtained if the speed of drying is controlled by
placing slides on cold, damp paper towels.
C. DNA Fibre Spreads
Many important aspects of the biology of replication
clusters, as well as fundamental features of the
replication process, can be studied using appropriately
labelled DNA fibre spreads
(Jackson and Pombo, 1998;
Takebayashi et al.
- Prepare PBS in distilled water.
- Prepare lysis mix with 0.5% (w/v) SDS, 200mM Tris-HCl (pH 7.4), and 50mM EDTA in distilled
- Prepare 3:1 (v/v) methanol:acetic acid fixative.
D. Immunolabeling Procedures
- Harvest cells labelled with the appropriate DNA
precursor analogue (or analogues for multiple
labelling) and resuspend in PBS at 106 cells/ml.
- Take a clean glass slide and place 2µl of the cell
suspension on the slide, about 2 cm from one end.
Leave the sample to stand for about 10min so that
the cells settle on to the glass surface; do not allow
the sample to dry completely.
- Add 5 µl of lysis mix to the cell sample and let stand
for 5 min at room temperature to lyse the cells.
- Tilt the slide to a ~20° angle so that the cell lysate
flows as a uniform smear to the bottom of the slide
(the smear will be about 4cm).
- Lie the slide on a flat surface and allow the spread
sample to dry.
- Fix the sample by immersing the slide in
methanol/acetic acid (3:1) for 5 min.
- Remove slides from the fixative and allow them to
dry. The dried slides can be stored in a fridge for
many days before immunolabelling.
Cell structures with incorporated DNA precursor
analogues must be stabilised by fixation prior to
immunolabeling. For light microscopy techniques,
paraformaldehyde is the fixative of choice. Samples
treated with methanol/acetic acid can be stained
directly, although for some applications it is also
advantageous to postfix samples with paraformaldehye.
Fixation and immunolabeling procedures used
for electron microscopy are specialised and beyond the
scope of this article. For details of these techniques, see
Hozák et al.
1. Cells on Glass
- Prepare PBS or physiological buffer (PB; see earlier
discussion) in distilled water.
- Prepare 4% paraformaldehyde in PBS or PB.
- Prepare 0.25% Triton X-100 in PBS or PB.
2. Encapsulated Cells
- After rinsing in PBS, place coverslips or slides in
4% paraformaldehyde for 10min at room temperature.
Permeabilised cells labelled in vitro should also be
fixed in this way but must be first washed in buffer to
remove unincorporated precursors.
- Wash samples twice with PBS.
- Permeabilise cells by incubation in 0.25% Triton
X-100 in PBS for 2min at room temperaturemthis
improves subsequent access of the antibodies.
- Wash gently in PBS (three changes over 5 min).
Samples can now be labeled with antibodies.
3. Antibody Binding
- After labelling in vitro, remove unincorporated precursors
by washing beads three times in 10 volumes
ice-cold PB for 15 min.
- Permeabilise by incubating in 0.25% Triton X-100 in
PB for 10 min at 0°C.
- Wash three times in 10 volumes ice-cold PB.
- Fix in ice-cold 4% paraformaldehyde in PB for
- Wash three times in 10 volumes ice-cold PBS.
Samples can now be labelled with antibodies as
Commercial anti-BrdU antibodies (Table II) react
poorly with BrdUMP in native DNA. Binding is
increased 10- to 20-fold if DNA is first denatured.
i. Acid Denaturation
- Prepare 2M HCl by diluting concentrated acid in
- Prepare 0.1M Na2B4O7 dissolved in distilled water.
- PBS/BSA/Tween is PBS containing 0.5% BSA and
0.1% Tween 20.
Comments and Pitfalls
- Rinse slides or coverslips in distilled water.
- Denature DNA in 2M HCl for 1 h at room
- Rinse five times in 0.1M Na2B4O7 to remove acid.
- Rinse twice in PBS/BSA/Tween and incubate
samples with PBS/BSA/Tween for 1-2h in a humid
atmosphere at room temperature to block nonspecificbinding
- Add first antibody, cover sample with a few
drops of PBS/BSA/Tween and 1/50 to 1/500 dilution
(determine empirically) anti-BrdU antibody, and
incubate 1-2h in a humid atmosphere at room
- Wash five times in PBS/BSA/Tween at room
temperature for 1 h.
- Add second antibody. Cover the sample with a
few drops of PBS/BSA/Tween and 1/500 to 1/1000
dilution of the labelled second antibody (Table II) and
incubate 1-2h in a humid atmosphere at room temperature.
The second antibody should be selected to
react with antibodies from the species in which the first
antibody was developed (Table II).
- Wash five times in PBS/BSA/Tween at room
temperature for 1 h.
- Prepare samples for microscopy using an appropriate
mountant (e.g., Vectashield) and seal coverslips
with nail varnish.
- Inspect samples using a suitable microscope.
Fluorochrome-coupled second antibodies give high
resolution and can be analyzed using confocalscanning
light microscopes and sensitive chargecoupled
device (CCD) cameras. Fluorescence-based
detection systems also allow convenient multiple
labelling (e.g., Aten et al.
, 1992; Jackson and Pombo,
1998; Schermelleh et al.
, 2001). As an alternative, it is
also possible to detect the location of the incorporated
precursor using enzyme-coupled second antibodies
(such as alkaline phosphatase). This approach is used
in routine histology of tissue sections but generally
offers lower resolution.
ii. Nuclease-Dependent Denaturation.
is accompanied by some loss of morphology. If
morphological considerations are critical, nucleasedependent
detection systems are preferred.
PBS/BSA/Tween is PBS containing 0.5% BSA and
0.1% Tween 20.
iii. Encapsulated Cells
- Rinse slides or coverslips once in PBS, twice in PBS/
BSA/Tween, and incubate in PBS/BSA/Tween for
1 h at room temperature.
- Add first antibody, cover samples with a few drops
of PBS/BSA/Tween, 1/50 to 1/500 dilution anti-
BrdU antibody, and 50µg/ml DNase I (Sigma), and
incubate 1-2 h in a humid atmosphere at 37°C.
- Perform steps 6-11 as following acid denaturation
- PBS/BSA/Tween is PBS containing 0.5% BSA and
0.1% Tween 20.
- 0.02mg/ml DAPI in sterile distilled watermthis is
a 1000× stock.
E. Patterns of DNA Synthesis
- Wash beads containing fixed cells twice in icecold
PBS/BSA/Tween and incubate in PBS/BSA/
Tween for 1 h at 0°C.
- Add first antibody, mix 100µl beads with 400µl
PBS/BSA/Tween containing 1/50 to 1/500 dilution of
appropriate first antibody, and incubate at 0°C for 2h
with periodic mixing.
- Wash three times in 10 volumes ice-cold PBS/
BSA/Tween for 30 min.
- Add second antibody, mix the bead pellet with
400µl PBS/BSA/Tween containing 1/500 dilution of
appropriate fluorochrome-coupled second antibody
(Table II), and incubate at 0°C for 2h with periodic
- Wash three times in 10 volumes ice-cold PBS/
BSA/Tween for 30min.
- Wash three times in 10 volumes PBS at room temperature
with 5min between washes; add 0.02µg/ml
DAPI or 1/250 dilution TOTO III iodide (fluorescence
in the far red spectrum) to second wash.
- Mount by mixing 5µl beads with an equal
volume of mounting medium (Vectashield), apply
coverslip with gentle pressure to eliminate excess
fluid, and seal with nail varnish.
- Inspect samples using a suitable microscope.
The techniques detailed earlier allow visualisation
of labeled DNA foci. These foci can be analysed by
indirect immunolabeling either immediately after
incorporation or many hours later. If the precursor
analogue (e.g., BrdU) is added to cells for <30 min and
indirect immunolabeling is performed immediately,
most labeled sites will correspond with sites of
ongoing DNA synthesis. This can be confirmed by
labeling DNA foci (as described earlier) in conjunction
with a marker for the replication factory. Antibodies
to PCNA are generally used for this purpose. Cells
labeled either in vivo
(Nakamura et al.
, 1986) or in vitro
(Nakayasu and Berezney, 1989) to reveal the sites of
nascent DNA synthesis display a few hundred discrete
nuclear sites, with patterns that are characteristic
of different stages of S phase (O'Keefe et al.
Humbert and Usson, 1992). Foci labeled after very
short incubations are associated with massive protein
complexes (Hozák et al.
, 1993) where many replicons
are duplicated together.
1. Labeling Sites of Ongoing DNA Synthesis
While nascent sites of DNA synthesis can be
labeled in living cells using short pulse labels of
appropriate analogues (e.g., BrdU), for technical
reasons it is preferable to label nascent sites in vivo
using permeabilised cells. The major advantages of in vitro
labeling are that precursors dNTP (e.g.,
biotin-dUTP) can be used directly and that soluble
pools of unassembled replication proteins are lost
during cell lysis.
- Physiological buffer as detailed earlier.
- 0.2% (w/v) saponin in PB-this is a 20× stock lysis
- 10× IM mix as indicated earlier supplemented with
20 µM biotin-dUTP.
- 4% paraformaldehyde in PB.
Comments and Pitfalls
- Permeabilise cells growing on coverslips or
encapsulated in agarose microbeads and label for
15-30min with a suitable labelled precursor analogue
as described earlier. Biotin-dUTP is recommended for
this application, although it may be convenient to label
the nascent DNA directly using a fluorescent precursor
analogue (Table I).
- If biotin-dUTP is used, wash samples three times
in PB to remove the unincorporated precursor and
fix the samples in 4% paraformaldehyde in PB as
- Visualise sites of incorporation as described
earlier, using an antibody to biotin (Table II).
- The engaged replication machinery can be visualised
in the same samples using an antibody to a
protein component such as PCNA using the standard
indirect immunolabelling technique: in PBS/BSA/
Tween add 1/500 dilution of the first anti-PCNA antibody
for 1-2h, wash with PBS/BSA/Tween, and then
apply appropriate fluorescently labelled second antibodies
in PBS/BSA/Tween as described earlier. Wash
the samples and mount as described earlier.
PCNA antibodies can be used to visualise assembled
replication proteins. The antibody from Alpha
Laboratories is a human autoimmune serum and can
be applied to samples directly. PC10, a mouse monoclonal
antibody, reacts poorly with PCNA at replication
sites fixed in paraformaldehyde. To use this
reagent, samples should be fixed by incubating for
10 min in methanol at -20°C.
F. Chromosome Dynamics and Live Cell
Many directly labeled fluorescent analogues of
DNA synthesis precursors can be used to visualise replication foci in living cells
Ansorge, 1995; Zink et al.
, 1998; Manders et al.
However, one limitation of this approach arises from
the inability of these charged molecules to cross the cell
membrane. Many techniques have been evaluated to
address this problem. Microinjection (Pepperkok and Ansorge, 1995; Zink et al.
, 1998) is an obvious possibility
but this is technically tedious and only ideally
suitable for labelling small numbers of cells. Other
alternatives include bead loading (Manders et al.
scratch loading (Schermelleh et al.
, 2001) and the use
of synthetic carrier complexes.
1. Scratch Loading
- Culture medium-as required for the specific cell
type under study.
- Culture medium supplemented with 10-20 µM dNTP analogue (Table I).
- Cells should be 50-75% confluent and growing on
glass coverslips or culture dishes with glass inserts.
- Remove the medium and apply medium with fluorescent
analogue -use 10 µl for each 13 mm diameter
and proportionally more if larger samples are
- Use the tip of a fine hypodermic needle to scratch
a series of parallel lines across the coverslip surface
-it is convenient to use lines separated by about
- After 1 min, add 0.5 ml of prewarmed medium and
let stand in incubator for 30min.
- Finally, wash in medium to remove any unincorporated
precursors, add medium to normal growth
conditions, and return culture dish to a humidified
incubator until use.
Carrier-mediated delivery of fluorescent nucleotides
(Table I) can be performed as follows.
- For each coverslip, prepare 3µl of the transfection
reagent FuGENE 6 mixed with 12µl PBS and incubate
at 4°C for 5 min.
- Add 1.5 µl of the desired fluorescent nucleotide analogue,
mix, and incubate at 4°C for 20min.
- Grow cells on 13-mm coverslips coated with
poly-L-lysine as described earlier.
- Pipette the fluorescent nucleotide carrier
complex (16.5 µl) onto a piece of parafilm and place the
coverslip (cell face down) on the droplet for 15min
- Rinse the cells in full medium, replace with fresh
medium, and return samples to an incubator prior to
analysis or further labelling treatments.
- If further analogues are to be used, simply repeat
the procedure at the desired time interval. Note,
however, that repeated applications do influence cell
viability, which is particularly pronounced if two
or more analogues are added consecutively without
allowing cells time to recover. Good results are obtained
if the separation between applications is at least 2 h.
- Samples can be inspected directly following
incorporation; using this technique, fluorescent
nucleotides introduced into an S-phase cell are consumed
in ~30min. For live cell experiments, transfer
the labelled cells into culture dishes with poly-L-lysine-coated glass coverslip inserts. Culture dishes
with gridded coverslips are also of use for relocating
individual cells during long-term analysis.
- Inspect samples using a convenient microscope
adapted for live cell imaging.
In living cells, studies of the dynamics of DNA foci
and their association with replication machinery can
be performed by labelling DNA foci in cells that have
a component of the replication machinery, such as
PCNA, tagged with GFP (Leonhardt et al.
G. Cell Cycle Analysis
In vertebrates, important features of the replication
process have been revealed using cell populations that
have been synchronised at critical points of the cell
cycle. Two points in the cycle are generally amenable
to cell synchronisation
. First, reagents that disrupt
microtuble function (e.g., nocodazole or colcemid)
allow cells to accumulate in mitosis. Cells that generally
grow as adherent monolayers can be purified in
mitosis using simple shake-off techniques that dislodge
only mitotic cells. Many compounds can be used
to accumulate cells at or close to the beginning of S
phase. Aphidicolin is the reagent of choice, as inhibition
is readily reversible and low concentrations added
to medium specifically inhibit the elongation phase of
DNA synthesis. The following protocol was used to
demonstrate the efficiency with which human replicons
are activated in different cell cycles (Jackson and
- Full medium, as required by cells under study -
serum should be added to support optimal growth.
- 250mM thymidine in sterile distilled water - this is
a 100× stock solution.
- 50µg/ml nocodazole in sterile distilled water-this
is a 1000× stock solution.
- 5mg/ml aphidicolin in DMSO - this is a 1000× stock solution.
- 10-100× stock solution of required DNA synthesis
precursor analogue (Table I) in medium.
Comments and Pitfalls
- Synchronise cells in mitosis by incubating
sequentially in medium supplemented with (i) 2.5 mM thymidine for 24 h; (ii) no additive for 12 h; (iii) 2.5 mM thymidine for 12h; (iv) no additive for 10h; and (v)
50ng/ml nocodazole for 4h. To remove inhibitors,
remove medium, wash cells once in fresh medium for
5 min, and replace with fresh medium.
- Wash cells to remove nocodazole and incubate in
fresh medium for 4-7h.
- Add 5µg/ml aphidicolin for 2-3 h.
- Wash cells in fresh medium supplemented with
a suitable precursor analogue (e.g., BrdU) and incubate
for 15-30 minutes as described previously. This will
pulse-label DNA foci in cells accumulated at the onset
of S phase.
- Return cells to fresh full medium.
- After a specified time, typically 1-5h, samples
can be pulse labelled further in medium supplemented
with a second precursor analogue (e.g., IdU).
- Alternatively, from 12h to many days later, steps
1-5 can be repeated using a second precursor analogue
- Indirect immunodetection of the two incorporated
analogues can now be performed on fixed cells
or spread DNA fibres as detailed earlier.
Timings must be determined empirically, as different
cell lines each have characteristic cell cycle parameters.
Trial experiments should be performed to
establish the time interval between releasing cells from
mitosis and the onset of S phase. For most mammalian
cell lines, cells synchronised in mitosis enter S phase
after 5-10h. Because G1 is the most variable period
of the cell cycle, additional aphidicolin treatment is
required to accumulate cells at the very beginning of S
phase. Conditions should be used that give 20-50%
cells undergoing replication.
The same approach can be applied using directly
labelled analogues for live cell imaging.
Using a combination of pulse labelling and fluorescent in situ
hybridisation (FISH) on DNA fibres, it is
also possible to analyse where DNA synthesis initiates
at specific chromosomal loci (Takebayashi et al.
H. Reconstituting Replication Sites
The protocols detailed earlier have been developed
to visualise DNA foci and sites of nascent DNA synthesis
in intact or permeabilised cells. For some applications it is convenient to extend our knowledge of the
replication process using systems that are amenable to
manipulation in vitro
. While it is beyond the scope of
this article to cover these applications in detail, the
authors would like to comment on their potential
merits. The best system in vertebrates takes advantage
of the ability of Xenopus laevis
egg extracts to assemble
nuclei when incubated with a suitable DNA (commonly
from sperm). Reconstituted nuclei are formed
that perform a single but complete round of DNA
synthesis, which begins about 30min after mixing.
Importantly, as the extracts can be manipulated by
the removal or addition of replication or cell cycle
components, this provides an excellent opportunity to
study pathways of activation and assembly of the
replication machinery. Mammalian nuclei cannot be
assembled from basic components in the same way,
although it is possible to manipulate assembly of the
replication machinery using permeabilised cells from
the late G1 phase of the cell cycle mixed with extracts
derived from S-phase cells.
Aten, J. A., Bakker, P. J., Stap, J., Boschman, G. A., and Veenhof, C.
H. (1992). DNA double labelling with IdUrd and CldUrd for
spatial and temporal analysis of cell proliferation and DNA replication. Histochem. J
Chong, J. P. J., Th6mmes, P., Rowles, A., Mahbubani, H. M., and
Blow, J. J. (1997). Characterisation of the Xenopus replication
licensing system. Methods Enzymol
Cremer, T., and Cremer, C. (2001). Chromosome territories, nuclear
architecture and gene regulation in mammalian cells. Nature Rev.
Dolbeare, F. (1995). Bromodeoxyuridine: A diagnostic tool in biology
and medicine. 1. Historical perspectives, histochemical methods
and cell kinetics. Histochem. J
Gratzner, H. G. (1982). Monoclonal antibody to 5-bromo and 5-
iododeoxyuridine: A new reagent for detection of DNA
replication. Science 218
Hozák, P., Hassan, A. B., Jackson, D. A., and Cook, P. R. (1993). Visualization
of replication factories attached to a nucleoskeleton. Cell 73
Hozák, P., Jackson, D. A., and Cook, P. R. (1994). Replication factories
and nuclear bodies: The ultrastructural characterization of
replication sites during the cell cycle. J. Cell Sci
Humbert, C., and Usson, Y. (1992). Eukaryotic DNA replication is a
topographically ordered process. Cytometry 13
Jackson, D. A., and Cook, P. R. (1985). A general method for preparing
chromatin containing intact DNA. EMBO J
Jackson, D. A., and Pombo, A. (1998). Replicon clusters are stable
units of chromosome structure: Evidence that nuclear organization
contributes to the efficient activation and propagation of Sphase
in human cells. J. Cell Biol
Krude, T., Jackman, M., Pines, J., and Laskey, R. A. (1997).
Cyclin/Cdk-dependent initiation of DNA replication in a human
cell-free system. Cell 88
Leonhardt, H., Rahn, H. P., Weinzierl, P., Sporbert, A., Cremer, T.,
Zink, D., and Cardoso, M. C. (2000). Dynamics of DNA replication
factories in living cells. J. Cell Biol
Ma, H., Samarabandu, J., Devdhar, R. S., Acharya, R., Cheng, P. C.,
Meng, C. L., and Berezney, R. (1998). Spatial and temporal
dynamics of DNA replication sites in mammalian cells. J. Cell
Manders, E. M. M., Kimura, H., and Cook, P. R. (1999). Direct
imaging of DNA in living cells reveals the dynamics of chromosome
formation. J. Cell Biol
Nakamura, H., Morita, T., and Sato, C. (1986). Structural organisation
of replicon domains during DNA synthesis phase in the
mammalian nucleus. Exp. Cell Res
Nakayasu, H., and Berezney, R. (1989). Mapping replication sites in
the eukaryotic cell nucleus. J. Cell Biol
O'Keefe, R. T., Henderson, S. C., and Spector, D. L. (1992). Dynamic
organization of DNA replication in mammalian cell nuclei: Spatially
and temporally defined replication of chromosome-specific
?-satellite sequences. J. Cell Biol
Pepperkok, R., and Ansorge, W. (1995). Direct visualization of DNAreplication
sites in living cells by microinjection of fluoresceinconjugated
dUTPs. Methods Mol. Cell. Biol
Schermelleh, L., Solovei, I., Zink, D., and Cremer, T. (2001). Twocolor
fluorescence labeling of early and mid-to-late replicating
chromatin in living cells. Chromosome Res
Takebayashi, S. I., Manders, E. M. M., Kimura, H., Taguchi, H., and
Okumura, K. (2001). Mapping sites where replication initiates
in mammalian cells using DNA fibers. Exp. Cell Res
Zink, D., Cremer, T., Saffrich, R., Fischer, R., Trendelenburg, M. E,
Ansorge, W., and Stelzer, E.H.K. (1998). Structure and dynamics
of human interphase chromosome territories in vivo
. Hum. Genet