Chromatin Assembly System
Chromatin is a periodic structure made up of
repeating, regularly spaced subunits, the nucleosomes.
Each nucleosome includes a core octamer of two molecules
each of histone proteins H3, H4, H2A, and H2B,
around which the DNA is wrapped almost twice.
The assembly and structure of chromatin modulate
the accessibility of proteins to the genome and hence
regulate all processes that utilize the DNA. Analyses
of transcription, DNA replication, and repair from in
vitro-assembled chromatin templates reveal a wealth
of information not provided by studies on naked DNA
templates, as the chromatinized template more accurately
reflects the state of the genome in the cell. This
article outlines and compares systems for the assembly
of recombinant DNA templates into arrays of
nucleosomes in vitro
to enable the reader to select the
appropriate chromatin assembly system for their
experimental question and resources. In addition, this
article describes the DNA replication-coupled chromatin
assembly system used in our laboratory and
directs the reader to recent method chapters providing
excellent descriptions of the other chromatin assembly
systems. Chromatin assembly systems fall into two
classes: "defined" systems where all the components
are purified or recombinant and "cell-free" systems
where the components required for assembly are
supplied by a crude extract. The cell-free chromatin
assembly systems can be further subcategorized into
replication-independent systems and DNA synthesiscoupled
systems. Defined chromatin assembly
systems should be used if the experiment requires that
any component of the chromatin assembly reaction or
the resulting chromatin be manipulated, such as the
nature of the histone proteins. Defined chromatin
assembly systems fall into two classes: histone transfer
systems and ATP-dependent chromatin assembly
A. Defined Chromatin Assembly Systems for
The core of the chromatin assembly reaction is the
transfer of histone proteins to DNA, as mediated by a
histone transfer vehicle or histone chaperone. If DNA
and histones are combined in the absence of a histone
transfer vehicle at physiological salt conditions, an
undefined insoluble aggregate is obtained due to the
intrinsic electrostatic attraction between the DNA and
the highly basic histone proteins. A histone transfer
vehicle binds to the histones and facilitates the ordered
formation of nucleosomes from histones and DNA by
reducing their inherent affinity for each other.
A variety of histone transfer vehicles can be used in vitro
to assemble chromatin, including polyanions
such as polyglutamate and counterions such as
sodium chloride. The observation that high salt concentrations
NaCl) dissociate nucleosomes led to the
discovery that this process was reversible by the progressive
dialysis of mixtures of DNA and histones
from high to low salt (Fig. 1A) (Lee and Narlikar,
2002). Chromatin assembly by salt dialysis is the
simplest and cleanest way to form nucleosomes and
has been invaluable for structural analyses of single
nucleosomes (mononucleosomes; Luger et al.
However, arrays of nucleosomes made by salt dialysis
are very close packed, with a nucleosome repeat length
of around 150-160bp as compared to that of 180-
190bp for nucleosomes in vivo
(nucleosome repeat length is the center-to-center distance between adjacent
nucleosomes; Fig. 1). This unphysiological closepacked
characteristic of the salt dialysed chromatin can
be overcome by incubation with an ATP-dependent
chromatin remodeling factor (see later; Fig. 1C). Salt
dialysis can yield nucleosomal arrays with more physiological
spacing when DNA templates that comprise
tandem arrays of nucleosome positioning sequences
derived from the sea urchin 5S RNA gene are used (Carruthers et al.
, 1999). However, the use of nucleosome
positioning sequences places a sequence limitation on
the DNA template and does not reflect the situation in
vivo where nucleosomes are generally not positioned by
the underlying DNA sequence.
Histone transfer in vitro
can also be achieved by
physiologically relevant histone chaperones, such as
nucleosome assembly protein 1 (NAP-l), chromatin
assembly factor 1 (CAF-1), and antisilencing function
1 (ASF1) or by histone storage proteins such as
nucleoplasmin and N1/N2. Histone chaperonemediated
chromatin assembly (in the absence of an
ATP-dependent chromatin remodeling factor) generally
results in the unphysiological random spacing of nucleosomes along the DNA (Fig. 1B). The major
applications for nucleosomal arrays assembled by
histone transfer are for protein interaction analyses
and for testing the ability of chromatin remodeling
factors to convert irregularly spaced or close-packed
nucleosomes into physiologically spaced, regular
B. Defined ATP-Dependent Chromatin
|FIGURE 1 Overview of chromatin assembly systems. Histones
can be deposited onto DNA to form nucleosomes in vitro via (A) the
sequential reduction in the salt (NaCl) concentration via dialysis,
resulting in regular, but close packed arrays of nucleosomes, or
(B) histone chaperone-mediated histone deposition onto DNA,
resulting in irregular arrays of nucleosomes. (C) The presence of an
ATP-dependent chromatin remodeling activity, either during or
after histone deposition, will result in regular, physiological spacing
of the nucleosomes.
In order to generate regularly spaced arrays of
nucleosomes that resemble chromatin in vivo
, an ATPdependent
chromatin remodeling factor is required in
addition to a histone chaperone (Fig. 1C). Chromatin
remodeling factors, e.g., ATP-utilizing chromatin
assembly and remodeling factor (ACF) and chromatin
accessibility complex (CHRAC), are multisubunit
protein complexes that use the energy from ATP
hydrolysis to space nucleosomes along the DNA
template (Fig. 1C). These ATP-dependent chromatin
assembly factors were identified by fractionation of Drosophila
embryo chromatin assembly extracts and
function together with histone chaperones, such as
NAP-1 or CAF-1 (Fyodorov and Levenstein, 2002). A
human ATP-dependent chromatin assembly factor,
termed remodeling and spacing factor (RSF), appears
to act as both the histone chaperone and the ATPdependent
chromatin remodeling factor during chromatin
assembly in vitro
(Loyola et al.
, 2001). These
defined ATP-dependent chromatin assembly systems
provide powerful approaches to generate regular
nucleosomal arrays with physiological nucleosomal
spacing, albeit at the expense of time-consuming
purification of the chromatin assembly factors and
C. Generation of Reagents for Defined
Chromatin Assembly Systems
In order to use defined chromatin assembly
systems, it is necessary to use purified or recombinant
chromatin assembly factors and histones. Recombinant
histone chaperones and ATP-dependent chromatin
remodeling factors are generated by affinity
purification following baculovirus-mediated expression
in Sf9 cells (Fyodorov and Levenstein, 2002). Core
histones (H3, H4, H2A, and H2B) can be purified from
a range of organisms using established protocols
(Schnitzler, 2002). In addition, recombinant Xenopus
, and yeast core histones can be expressed
and purified from Escherichia coli, allowing assembly
of chromatin lacking posttranslational modifications or with specific histone mutations (Lee and Narlikar,
2002; Levenstein and Kadonaga, 2002; Luger et al.
1999; White et al.
, 2001). For those with time constraints,
individual recombinant Xenopus
can be purchased from Upstate Biologicals and individual
purified core histones from calf thymus can be
purchased from Roche Applied Science. Due to the
highly conserved nature of chromatin assembly factors
and nucleosome structure across eukaryotes, it is not
generally necessary to adhere to species specificity
within the chromatin assembly machinery, histones,
and the subsequent application of the chromatin
templates. For example, there is no gross functional
or structural difference in the chromatin assembled
from yeast, human, or Drosophila
core histones using
, or yeast chromatin assembly
D. Cell-Free Replication-Independent
Chromatin Assembly Systems
Cell-free chromatin assembly systems were developed
prior to the defined ATP-dependent chromatin
assembly systems, but are still the system of choice for
many researchers. Technically, chromatin assembly in
cell-free systems requires less empirical optimization
than defined ATP-dependent chromatin assembly systems, while still generating high-quality regular
arrays of nucleosomes with physiological spacing in
an ATP-dependent manner. Extracts remain a requirement
for the analysis of processes coupled to chromatin
assembly, such as the assembly of chromatin
during ongoing DNA synthesis in vitro
. Limitations of
cell-free chromatin assembly systems include the
inability to fully control the nature of histones and
nonhistone proteins incorporated into the chromatin
or the chromatin assembly factors used for assembly,
as these are contained within the cell-free extract.
Conversely, the crude nature of these extracts can be
an advantage if, for example, unknown factors are
required to facilitate subsequent transcriptional activation
from the chromatin template.
Cell-free extracts for generating physiologically
spaced, regular arrays of nucleosomes have been
derived from Xenopus laevis
eggs and oocytes, Drosophila melanogaster
embryos, and human tissue
culture cells by high-speed (HS) centrifugation (Table
I) (Bonte and Becker, 1999; Fyodorov and Levenstein,
2002; Gruss, 1999; Tremethick, 1999). The realization
that the early developmental stages of Xenopus
contain stockpiles of the components necessary
to assemble the rapidly dividing DNA into chromatin
was key to the development of highly active
chromatin assembly extracts. For transcription analyses, the DNA template is usually assembled into chromatin
using these extracts, followed by the addition of
transcription factors or a transcription extract. Chromatin
templates generated from the Xenopus
extract are inherently flawed for subsequent transcriptional
analyses due to the abundance of variant linker
histone B4 and transcription factors in the extract. In
fact, assembly of transcription complexes competes
with chromatin assembly in the Xenopus
extract. In general, the Xenopus
egg HS extract is used
for DNA synthesis-coupled chromatin assembly,
whereas the Xenopus
oocyte extract (S150; where S
refers to the sedimentation coefficient) is used for
assembling templates into chromatin for their subsequent
transcriptional analyses (Gaillard et al.
Tremethick, 1999). The Drosophila
S150 and S190
embryo extracts (Bonte and Becker, 1999; Fyodorov
and Levenstein, 2002) have been widely used to
assemble chromatin templates for subsequent analyses
of transcriptional regulation, recreating many of the
features of transcriptional regulation in vivo
and Kadonaga, 1998). These extracts also contain
factors that can modify subsequent transcription reactions,
such as chromatin remodeling activities, and
therefore it may be desirable to purify the chromatin
templates by sedimentation centrifugation or size
exclusion prior to transcription analysis. Removal of
nonhistone proteins from the chromatin may be
achieved by treatment with Sarkosyl prior to purification
of the chromatin template (Mizuguchi and Wu,
1999). However, defined chromatin assembly systems
are the ideal method for complete control of the subsequent
transcription reaction on chromatin templates.
A chromatin assembly extract has been developed
from Saccharomyces cerevisiae
that enables the powerful
combination of genetics and biochemistry (Table I).
This yeast extract also supports transcriptional activation
and can be isolated from any yeast strain that has
been altered by genetic manipulations. This enables
investigation of the effect of specific mutations on
chromatin assembly and transcriptional regulation of
the in vitro
-assembled chromatin templates (Schultz,
1999). However, this extract is limited by its inability
to generate extensive regular arrays of nucleosomes
E. DNA Synthesis-Coupled Chromatin
In the cell, the majority of chromatin assembly is
coupled to ongoing DNA replication and repair. The
process of chromatin assembly coupled to doublestrand
DNA replication has been recreated in vitro
using extracts from human tissue culture cells (Table
I). In addition, Drosophila
, human cell, and Xenopus
HS extracts can mediate nucleotide excision repaircoupled
chromatin assembly on damaged templates
and second-strand synthesis-coupled chromatin
assembly on M13 templates (Bonte and Becker, 1999;
Gaillard et al.
, 1999). The human 293 cell extract has
been widely used to study the process of DNA replication
coupled-chromatin assembly using an SV40
origin to initiate replication via the method described
in this article (Fig. 2). In this system, DNA replication
occurs on only a portion of the total DNA templates,
allowing comparison of chromatin assembly on the
newly replicated DNA (labelled with a radioactive
nucleotide) to chromatin assembly on the total DNA
(Fig. 2). As such, this system has led to the identification
and characterization of histone chaperones that
mediate chromatin assembly onto newly synthesized
DNA, such as CAF-1 and ASF1 (Smith and Stillman,
1989; Tyler et al.
, 1999). Furthermore, the SV40 DNA
template can be preassembled into chromatin using Drosophila
chromatin assembly extracts
and then replicated using human DNA replication
extracts (Gruss, 1999). Refinement of this approach
may recreate epigenetic inheritance of chromatin structures in vitro
F. Evaluation of in Vitro.Assembled
|FIGURE 2 DNA replication-coupled chromatin assembly. (A) Overview of the SV40 T-antigen-driven
replication assay in a human cell-free extract coupled to chromatin assembly. The newly replicated DNA (red)
becomes labelled by the incorporation of a radioactive nucleotide. (B) Example of supercoiling analysis of
the products of DNA replication-coupled chromatin assembly. By comparison of EtBr staining and autoradiogram
of the same agarose gel, preferential assembly (supercoiling) of the replicated DNA into chromatin
is apparent upon addition of CAF-1.
Once assembled, it is important to check the extent
of nucleosome assembly and the regularity of nucleosomal
spacing. The extent of chromatin assembly can
be measured by DNA supercoiling analysis, as the formation
of each nucleosome introduces one negative
supercoil into covalently closed circular DNA. The
number of negative supercoils therefore corresponds
to the number of nucleosomes assembled and is
measured after deproteinization by agarose gel electrophoresis.
Plasmid supercoiling by an extract cannot
always be attributed to chromatin assembly, as these
extracts potentially contain unrelated DNA supercoiling
activities. Therefore, it is necessary to confirm that
the supercoiling is due to chromatin assembly, usually
by the micrococcal nuclease digestion assay. In this
assay, the chromatin is partially digested by micrococcal
nuclease, which cleaves both strands of DNA
between the nucleosomal cores. The resulting chromatin
fragments are deproteinized and the DNA is
resolved by agarose gel electrophoresis. A DNA ladder
derived from the chromatin fragments is observed
if the assembly was efficient and the nucleosomes
were regularly spaced, and the size of the DNA fragments allows the nucleosome repeat length to be
II. MATERIALS AND
Plasmid template carrying SV40 origin of DNA
replication [we use pSV011 (Stillman, 1986)]. SV40-T
antigen, affinity purified (Simanis and Lane, 1985)
or purchased (Cat. No. 5800-01) from CHIMERx.
Spinner-adapted version of HEK293 cells (Cat. No.
CRL1573) is from ATTC. Joklik's modification of MEM
(Cat. No. 1032324) and sodium butyrate (Cat. No.
218453) are from ICN. Fetal bovine serum (Cat. No.
16000-044) is from Life Technologies Inc. Ribonucleotides
(ATP, CTP, GTP, UTP, Cat. Nos. E6011, E6021, E6031, E6041) and deoxyribonucleotides (dATP, dCTP,
dGTP, dTTP, Cat. No. U1330) are from Promega. Filters
(0.22 µm) (Cat. No. GSWP04700) are from Millipore. A
40-ml Wheaton dounce homogenizer with B pestle
(Cat. No. 06-435-C), Corex 8445 tubes (Cat. No. 05-566-
55), agarose (Cat. No. BP1356), chloroform (Cat. No.
C298), EDTA (Cat. No. S311), dithiothreitol (DTT, Cat.
No. BP172-25), glycerol (Cat. No. G3320), HEPES (Cat.
No. BP310), magnesium chloride (MgCl2
, Cat. No.
M33), phenol: chloroform:isoamylalcohol 25:24:1
(Cat. No. BP17521), potassium chloride (KCl, Cat. No.
P217), potassium hydroxide pellets (KOH, Cat. No.
P250), potassium phospate monobasic (KH2
No. P285), proteinase K (Cat. No. BP1700), sodium
acetate trihydrate (NaOAc, Cat. No. S209), sodium
chloride (NaCl, Cat. No. S271), sodium phosphate
O Cat. No. S373), sodium phosphate
Cat. No. S369), Tris base (Cat. No. BP152), xylene cyanol FF (XC, Cat. No.
BP565), and X-ray film (Cat. No. 05-728-24) are from
Fisher. Boric acid (Cat. No. B0252), bromphenol blue
(BPB, Cat. No. B-6131), creatine phosphate (Cat. No.
P7936), creatine phosphokinase (CPK, Cat. No. C3755),
ethidium bromide (EtBr, Cat. No. E-8751), glycogen
(Cat. No. G0885), IGEPAL (NP40, Cat. No. I3021),
RNase A (Cat. No. R6513), and sodium hydroxide
pellets (NaOH, Cat. No. S-0899) are from Sigma.
Sodium dodecyl sulphate (SDS, Cat. No. 161-0302) is
from Bio-Rad. [α-32
P]dATP 3000Ci/mmol, 10µCi/µl
(Cat. No. BLU512H) is from Dupont NEN. Complete
protease inhibitor cocktail (Cat. No. 1836153) is from
A 37°C tissue culture incubator (CO2
and a magnetic stir plate for spinner flasks (Thermodyne
Cat. No. 45700) are from Fisher. The 6-liter
spinner flask (Cat. No. 1965-06000) is from Bellco.
Refrigerated centrifuge Sorvall RC-5B with SS34, HB6,
and GSA rotors (or equivalent). Beckman ultracentrifuge
with Ti70 rotor. Rotary Speed-Vac, gel dryer,
and -80°C freezer are from Thermo Savant (or similar).
Agarose gel electrophoresis equipment, Gibco H5
system (or similar). Liquid nitrogen tank. Eppendorf
Model 5415D microcentrifuge (or similar).
A. Preparation of DNA Replication-
Dependent Chromatin Assembly Extract
These procedures are modified from those of
- Phosphate-buffered saline (PBS): 136mM NaCl,
2.7 mM KCl, 3.4 mM Na2HPO4, and 1.47 mM KH2PO4.
For 100ml of 1X PBS, combine 90 ml MilliQ or doubledistilled
water (ddH2O) and 10ml 10X PBS, use within
days. For 1 liter of 10X PBS, to 900ml ddH2O add 80g
NaCl, 2g KCl, 9.2g Na2HPO4·7H2O, and 2g KH2PO4.
Stir until dissolved and then add ddH2O to 1-liter final
volume. Autoclave and store 10X PBS at 4°C.
- 1M HEPES-KOH, pH 8.0: For 1 liter, to 600ml
ddH2O add 238.3 g HEPES. Once dissolved, adjust pH
to 8.0 using KOH pellets and add ddH2O to 1-liter final
volume. Pass through 0.22-µm filters. Store in a sterile
bottle at 4°C.
- 3 M KCl: For 1 liter, to 700ml ddH2O add 223.68 g
KCl. Once dissolved, add ddH2O to 1-liter final volume.
Autoclave and store at room temperature.
- 1M MgCl2: For 1 liter, to 700ml ddH2O add
203.3g MgCl2. Once dissolved, add ddH2O to 1-liter
volume. Autoclave and store at room temperature.
- 0.5M DTT: For 50ml, dissolve 3.85g DTT in
50ml ddH2O and store at -20°C.
- 1M sodium phosphate, pH 7.2: Dissolve 26.807g
NaH2PO4 in 70ml ddH2O and adjust volume to 100ml
once dissolved. Dissolve 13.8 g Na2HPO4·7H2O in 80ml
ddH2O and adjust volume to 100ml once dissolved.
Combine the two solutions together (approximately
68 ml :31 ml, respectively) until pH 7.2 is reached. Store
at room temperature.
- 1M Na butyrate: 1M Na butyrate and 20mM sodium phosphate, pH 7.2. For 100ml, dissolve 11 g
butyric acid (sodium salt) in 80ml ddH2O and then
pH to 7.2 with NaOH. Next, add 2ml 1M sodium
phosphate, pH 7.2. Add ddH2O to 100-ml final
volume. Store at 4°C. This solution has an unpleasant
- Hypotonic buffer: 20mM HEPES-KOH, pH 8.0,
5mM KCl, 1.5mM MgCl2, and 0.1mM DTT. For
100ml, add 2ml 1M HEPES-KOH stock, 0.16ml 3M KCl, 150µl 1M MgCl2, and 20µl 0.5M DTT (stored at
-20°C. Bring to volume with ddH2O. Prepare fresh
and chill on ice.
- 5M NaCl: For 1 liter, to 700 ml ddH2O add 292.2 g
NaCl. Once dissolved, add ddH2O to 1-liter final
volume. Autoclave and store at room temperature.
B. DNA Replication-Dependent Chromatin
Assembly and Supercoiling Assay
- Grow 4 liters of 293 cells in Joklik's modification
of MEM plus 5% bovine calf serum in a 6-liter
spinner flask to 6 × 105 cells/ml at 37°C. Note that
because this medium is not buffered by sodium
bicarbonate, you can use a sealed spinner in a 37°C room if necessary.
- Spin down 293 cells in six GSA bottles at 2500rpm
at 4°C for 10min with brakes off. Pour off and
discard the supernatant.
- Repeat step 2 using the same GSA bottles until all
cells are pelleted. Keep the bottles on ice whenever
possible. Perform all the following procedures in a
- Resuspend cells in the liquid remaining in the GSA
bottles and transfer to an SS34 tube on ice. Wash
the remaining cells out of the GSA bottles with
chilled PBS and pool into the SS34 tube.
- Centrifuge at 2500rpm at 4°C for 3min and then
slowly pour off and discard the supernatant.
- Add 20ml of chilled PBS to the cell pellet and
resuspend by gentle pipetting with a plastic 25-ml
- Centrifuge cells again for 3 min and slowly pour
off and discard the supernatant.
- Estimate the volume of packed cells in each tube
in order to determine the volume of hypotonic
buffer to be added later (by pipetting water into
another SS34 tube and estimating the pellet
volume by comparison, usually approximately 7-
- To 100 ml hypotonic buffer, add 0.5 ml of 1M Na
butyrate (to 5mM final) and protease inhibitor
- Add 20ml of hypotonic buffer (with additives) to
cells and resuspend by gentle pipetting.
- Centrifuge at 2500 rpm at 4°C for 5 min and remove
and discard the supernatant using a pipette.
- Resuspend cells in 1 ml of hypotonic buffer for
each milliliter of packed cell volume estimated in
step 8. Resuspend cells by gentle pipetting. Incubate
cells on ice for 10min.
- Transfer cell suspension to a 40-ml dounce homogenizer.
Rinse SS34 tube with a small amount of
hypotonic buffer and pour the rinse into the
dounce homogenizer. Remove 10 µl of cell suspension
for step 14.
- Dounce cells on ice for 25-30 passages with a "B"
pestle. Remove 10µl of cell suspension, and
compare cells before and after douncing using an
optical microscope to estimate extent of lysis. If
there are significant numbers of intact cells after
douncing, dounce for a few more passages.
- Pour lysed cells into two prechilled 30-ml Corex
tubes. Incubate on ice for 30min.
- Centrifuge in an HB6 rotor at 10,000 rpm at 4°C for
10min. Pour the supernatant into a 15-ml conical
tube to estimate volume. Add NaCl to 0.1M final
concentration (a 1/50 dilution of 5M NaCl) into
the supernatant and mix gently.
- Divide solution between two ultracentrifuge tubes
(Beckman Ti70) and centrifuge in a prechilled Ti70
rotor at 31,000rpm at 4°C for 60min.
- Transfer the supernatant to a new tube (it is okay
if some lipids transfer over). Divide into 0.5-ml
aliquots, freeze in liquid N2, and store at -80°C.
- 1M creatine phosphate: Add 1 g of creatine phosphate
to 80 µl 1M HEPES-KOH, pH 8.0, and 3.92 ml
ddH2O. Store at -80°C.
- 5X RM: 200mM HEPES-KOH, pH 8.0, 15mM ATP, 1 mM CTP, 1 mM GTP, 1 mM UTP, 0.125 mM dATP, 0.5mM dCTP, 0.5mM dGTP, 0.5mM dTTP,
200 mM creatine phosphate, 40 mM MgCl2, and 2.5 mM DTT. To make 1.2ml 5X RM, combine, in order,
426.5 µl ddH2O, 240 µl 1M HEPES-KOH, pH 8.0, 180 µl
100mM ATP, 12µl 100mM CTP, 12µl 100mM GTP,
12 µl 100 mM UTP, 1.5 µl 100 mM dATP, 6 µl 100 mM dCTP, 6 µl 100 mM dGTP, 6 µl 100 mM of dTTP, 240 µl
1M creatine phosphate, 28 µl 1M Mg2Cl, and 6 µl 0.5M DTT. Freeze in liquid N2 and store at -80°C.
- 2M Tris-HCl, pH 7.5: to make 2 liters, add
484.4 g Tris base to 1.5 liters of ddH2O. Adjust pH to
7.5 with HCl. Pass through a 0.22-µm filter and store
in in sterile bottle at room temperature.
- CPK: 5mg/ml CPK, 50% glycerol, 25mM Tris-HCl, pH 7.5, 1 mM EDTA, 25 mM NaCl, and 1 mM DTT. To make 10ml CPK, add 50mg creatine phosphokinase
to 4.785 ml ddH2O, 5 ml glycerol, 125µl 2M
Tris-HCl, pH 7.5, 20µl 0.5M EDTA, 50µl 5M NaCl,
and 20µl 0.5M DTT. Make 10-µl aliquots, freeze in
liquid N2, and store at -80°C.
- Master mix: For each of x reactions (x = n + 1,
where n is the number of reactions being performed),
combine on ice in the following order: 5µl 5X RM,
0.1 µl CPK, 1 µl template DNA (at 50ng/µl), 1 µl SV40
T-antigen, and 0.1 µl [α-32P]dATP. Use immediately.
- 0.5M EDTA, pH 8.0: For 1 liter, to 700ml ddH2O
add 186.12g EDTA. Add NaOH pellets until white
emulsion begins to clear. Adjust pH to 8.0 with NaOH
pellets. Add ddH2O to 1-liter final volume. Autoclave
and store at room temperature.
- 25 mM buffer A: 18.75 mM Tris-HCl, pH 7.5, 2.5%
glycerol, 1 mM EDTA, and 0.125% NP-40. For 4ml,
combine 3.45 ml ddH2O, 37.5 µl 2M Tris-HCl, pH 7.5,
500 µl glycerol, 10 µl 0.5M EDTA, and 4 µl NP-40. Store
- 10 mg/ml RNase A: Dissolve 1 g RNase A in 100 ml
ddH2O. Make aliquots and store at -20°C.
- EDTA/RNase buffer: For 1 ml, combine 935µl
ddH2O, 50µl 0.5M EDTA, pH 8.0, and 15µl 10mg/ml
RNase A. Use immediately.
- Glycogen stop buffer: 20 mM EDTA, 1% (w/v)
SDS, 0.2M NaCl, and 250µg/ml glycogen. For 50ml,
combine 45.5 ml ddH2O, 2 ml 0.5M EDTA, 500 mg SDS,
2ml 5M NaCl, and 12.5mg glycogen. Store at room
- Proteinase K: 2.5mg/ml proteinase K, 10mM Tris-HCl, pH 7.5, and 1 mM EDTA. To make 10 ml, dissolve
25mg proteinase K in 9.9ml ddH2O, 50µl 2M
Tris-HCl, pH 7.5, and 20 µl 0.5M EDTA. Make aliquots
and store at -20°C.
- 3M NaOAc: For 1 liter, to 500ml ddH2O add
408.24g sodium acetate trihydrate. Once dissolved,
add ddH2O to 1-liter final volume. Autoclave and store
at room temperature.
- 1X TBE: 89mM Tris, 89mM boric acid, and
2.5 mM EDTA. For 1 liter of 1X TBE, combine 900ml ddH2O and 100ml of 10X TBE, For 1 liter 10X TBE, to
600ml ddH2O add 108 g Tris base, 55 g boric acid, and
7.49 g EDTA. Stir until dissolved and then add ddH2O
to 1-liter final volume. Store at room temperature.
Discard solution when white particles appear.
- 1X TBE gel-loading buffer: 89mM Tris, 89mM boric acid, 2.5 mM EDTA, 6% glycerol, 0.05% BPB, and
0.05% XC. For 50ml of 1X TBE gel loading buffer,
combine 15 ml ddH2O, 15 ml glycerol, 25 ml 10X TBE,
0.125g BPB, and 0.125g XC. Store at 4°C.
- 10mg/ml EtBr: Add 0.5g EtBr (weighed in a
fume hood) to 50ml ddH2O. Store in the dark at
- Thaw T-antigen, CPK, 293 extract, and samples
being tested in a room temperature water bath and
place on ice once thawed. Assemble master mix on ice.
Set up replication reactions on ice by adding in order
7.8 µl 25 mM buffer A, 7.2 µl master mix, and 10 µl 293
replication extract. Mix by pipetting up and down.
If samples are being tested for their effect on DNA
replication and/or chromatin assembly, add the
samples first and adjust the volume of 25 mM buffer A
so that the reaction has a final volume of 25 µl. Discard
remainder of CPK aliquot and master mix. Freeze all
unused reaction components in liquid N2 and store at
- Incubate replication reactions at 30°C for 90min.
- Stop reactions by the addition of 6.25µl
EDTA/RNase buffer. Incubate at room temperature for
- Add 95µl glycogen stop buffer and 5µl proteinase
K. Incubate at 37°C for 15 min.
- Extract samples with 1 volume of phenol:chloroform:
isoamyl alcohol (25:24:1) and then with 1
volume of chloroform:isoamyl alcohol (24:1).
- Precipitate DNA by addition of 1/10 volume of
3M NaOAc and 3 volumes of 100% EtOH. Invert to
mix and microcentrifuge at full speed for 15 min.
- Remove and discard supernatant and wash
pellets with 75% EtOH. Dry pellets in a Speed-Vac.
- Resuspend pellets in 8µl of 1X TBE gel loading
buffer. Load into wells of 1% agarose gel in 1X TBE and
run until BPB runs off the gel (35 V for 14 h).
- Stain gel in 500ml ddH2O + 30µl 10mg/ml
EtBr for 30min. Destain in ddH2O for 30-60min and
- Dry gel on two layers of Whatman 3 MM paper
overlaid with Saran wrap on a gel drier for 2h with
heater set to 65°C. Expose gel to film.
Figure 2B shows the products of the DNA replication-
coupled chromatin assembly reactions, where the top shows total DNA and the bottom shows the newly
- For each batch of T-antigen, the optimal amount
needs to be determined empirically by determining the
lowest amount that gives the maximum amount of
replication. Occasionally, the volume of each extract
for chromatin assembly may need to be determined in
a similar manner.
- The amount of sample to use in a typical reaction
needs to be determined empirically, as addition of too
much chromatin assembly factor (i.e., CAF-1, ASF1)
will inhibit DNA replication. Because too much salt in
the samples can interfere with replication efficiency, in
general it is advisable to dialyze samples into 25 mM buffer A before replication analysis.
- Controls for replication dependence, as opposed
to nucleotide excision repair of the nicked plasmid,
include omission of T-antigen or use of a plasmid
lacking the SV40 origin of DNA replication.
In our hands, there is significant variation between
different 293 extracts in terms of the amount of active
CAF-1 and ASF1 that they contain, for unknown
reasons. For example, we have generated extracts that
do not require the addition of CAF-1 for chromatin
assembly, only require the addition of CAF-1, or
require the addition of both CAF-1 and ASF1, and we
have even generated extracts that fail to replicate
DNA! Therefore, each extract needs to be assessed by
performing the replication reaction and supercoiling
analysis with and without complementation by CAF-
1, ASF1, or crude extracts. This extract variability is not
an issue if a crude extract (such as the cell-free chromatin
assembly extracts described in Section I,C) are
being used to supply chromatin assembly factors. In
fact, the variability in the extracts has allowed the
original biochemical identification of CAF-1 and ASF1
by complementation of the 293 extracts (Smith and
Stillman, 1989; Tyler et al.
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