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INTRODUCTION |
The replication of eukaryotic chromosomes is integrated into the
regulation of the cell division cycle (1). During S phase, replication
of the genomic DNA is tightly coupled to an assembly of the nascent DNA
into chromatin (2-5). Chromatin assembly not only allows packaging of
the genomic DNA into the nucleus but it is also involved in the
regulation of essential DNA transactions such as gene expression,
recombination, and repair. The fundamental repeated unit of chromatin
structure is the nucleosome core particle, which contains 146 base
pairs of DNA wrapped in 1.75 left-handed superhelical turns around a
core of two copies of each core histone protein H2A, H2B, H3, and H4
(6). Synthesis of histone proteins occurs during S phase and is coupled
to ongoing DNA replication (7).
The assembly of new nucleosomes during DNA replication can be analyzed
in cytosolic extracts from human somatic cells. Double stranded
circular DNA containing the simian virus 40 (SV40) replication origin
is able to replicate in a human cytosolic extract under the control of
the virally encoded initiator protein and DNA helicase, T antigen (8).
The cytosolic extract also catalyzes complementary DNA strand synthesis
in the absence of any viral factors on single-stranded DNA templates
(9). Nucleosome assembly on replicating DNA molecules in these systems
depends on the addition of the nuclear chromatin assembly factor 1 (CAF-1)1 to the reaction (9,
10). CAF-1 mediates deposition of histone H3/H4 tetramers on the DNA
from newly synthesized H3 and H4, which are present in the cytosolic
extract (11). In a second step independently of CAF-1, the nucleosome
core particles are completed by association of two H2A/H2B dimers to a
H3/H4 tetramer (11).
CAF-1 was purified from human cell nuclei as a trimeric protein complex
of the subunits p150, p60, and p48 (10), or as a larger complex termed
chromatin assembly complex (CAC), also containing newly synthesized
histones H3 and H4 (12). The two larger subunits p150 and p60 directly
interact with each other and both are essential for nucleosome assembly
during DNA replication (13). Immunoprecipitation experiments
demonstrated a direct interaction of the p150 subunit with newly
synthesized and modified histones H3 and H4 (13). The small subunit p48
binds to free, but not to nucleosomal histone H4 in the absence of the
p150 and p60 subunits (12, 14). These histone-binding properties
support a role for CAF-1 as a chaperone (15), targeting free histones
to replicating DNA for an assembly into nucleosomes. CAF-1 also
mediates nucleosome assembly during DNA repair synthesis following
UV-induced damage of the DNA templates (16-18). The chaperone model of
CAF-1 is supported by the non-cooperative and stoichiometric nucleosome
assembly mechanism during complementary DNA strand synthesis in
vitro (9).
CAF-1 is a prime candidate for nucleosome assembly during S phase in
somatic cells because of its substrate specificity for replicating DNA
templates (9, 10). However, nucleosome assembly by CAF-1 can be
experimentally uncoupled from ongoing DNA replication by adding CAF-1
to an in vitro reaction after DNA strand synthesis has been
inhibited by aphidicolin, suggesting that newly replicated DNA is
marked for subsequent nucleosome assembly by CAF-1 (19). This marking
depends on a replication factor, proliferating cell nuclear antigen
(PCNA) (20). During DNA replication, PCNA is reversibly loaded onto the
DNA by replication factor C, forming a topologically closed ring
structure around the duplex DNA strand (reviewed in Refs. 21-23).
Unloading of PCNA from replicated templates by replication factor C
prevents subsequent nucleosome assembly by CAF-1, and antibodies
against PCNA inhibit CAF-1-mediated chromatin assembly, suggesting that
the continued presence of PCNA on replicated templates is required for
CAF-1 activity (20). These results established a molecular link between
the replication fork and the nucleosome assembly machinery of CAF-1 via
PCNA. However, successful coupling of these two processes required
additional, as yet unknown factors present in the unfractionated
cytosolic extract (20).
We are addressing the identity of accessory factors required for
nucleosome assembly by CAF-1 during DNA replication using an
independent approach. During S phase, the two large subunits of CAF-1
co-localize with the intranuclear sites of DNA replication (24). The
p60 subunit of CAF-1 changes its phosphorylation state at key
regulatory transitions during the cell cycle. Coincident with a
recruitment to replication foci at the G1 to S phase
transition, p60 becomes partially dephosphorylated (25). In mitosis,
p60 becomes hyperphosphorylated, coinciding with loss of nucleosome assembly activity of CAF-1 and its displacement from chromatin (25).
These data suggest an involvement of reversible phosphorylation by cell
cycle-specific protein kinases and protein phosphatases in regulating
CAF-1 activity during the cell division cycle.
Key players of cell cycle control in somatic mammalian cells are
cyclins and cyclin-dependent, serine/threonine protein
kinases (Cdks) (reviewed in Refs. 26-33). The cyclin subunits
contribute to timing, substrate specificity, intracellular
localization, and binding to other regulatory proteins of the catalytic
Cdk subunits. Following mitogen stimulation, a cascade of Cdk
activities is required for timely triggering initiation of nuclear DNA
replication at the onset of S phase. A sequential synthesis of the
regulatory D-, E-, and A-type cyclins results in sequential binding to,
and activation of, the appropriate Cdk subunits. During G1
phase progression, D-type cyclins associate with Cdk4/6 and their
activity is involved in expression of S phase-specific genes.
Initiation of and progression through S phase is under control of
cyclin E/Cdk2 and cyclin A/Cdk2 activity. Following successful
replication, condensation of interphase chromatin into mitotic
chromosomes and their segregation into the two daughter cells is
controlled by B-type cyclins complexed to Cdk1. Phosphorylation by
active Cdks is reversible, and phosphates on serine and threonine
residues can be removed by protein phosphatase types 1 and 2 (32,
34).
In this paper, we investigate the functional requirement of reversible
protein phosphorylation for nucleosome assembly by CAF-1 during DNA
replication in human cell extracts. Specific inhibitors for
cyclin-dependent kinases and protein phosphatases inhibited
nucleosome assembly by CAF-1. These results establish a requirement for
reversible protein phosphorylation by G1/S phase-specific Cdks and protein phosphatase type 1 (PP1) for CAF-1 activity in vitro. Furthermore, the p60 subunit of CAF-1 is reversibly
phosphorylated by cyclin/Cdks and PP1 activities during the nucleosome
assembly reaction in vitro. These data provide a novel
molecular link between the control of chromatin assembly during DNA
synthesis and the cell cycle machinery.
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MATERIALS AND METHODS |
Cell Culture and Extract Preparation--
HeLa-S3 cells were
cultured as exponentially growing subconfluent monolayers on 145-mm
plates in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.), supplemented with 10% fetal calf serum (Life Technologies,
Inc.), 10 units/ml penicillin (Sigma), and 0.1 mg/ml streptomycin
(Sigma). Cytosolic extracts and CAF-1 were prepared as detailed
previously (9, 25).
DNA Synthesis and Nucleosome Assembly
Reactions--
Complementary DNA strand synthesis and nucleosome
assembly reactions were essentially performed as described (9), with the following modifications. Standard reactions contained cytosolic extract (150 µg of protein) from asynchronously proliferating cells,
unless indicated otherwise, and 30 ng of single-stranded M13mp18 DNA as
template. Semiconservative DNA replication of double-stranded DNA was
performed under identical conditions, but using 15 ng of
double-stranded M13O1 DNA containing the SV40 control region as
template (35) and 5 µg of purified SV40 large T antigen as initiator
protein (gift of C. Gruss, University of Konstanz, Konstanz, Germany)
(8). CAF-1 was isolated from interphase nuclei (9), or from mitotic
chromatin (25), and was added at 6 µg of total protein where indicated.
Stock solutions of roscovitine (50 mM), olomoucine (50 mM), okadaic acid (50 µM), and protein
phosphatase inhibitor 2 (45 µM; all Calbiochem) were
dissolved in Me2SO. Sodium vanadate was dissolved in
distilled H2O at 100 mM. The first 90 amino
acids from human p21 protein comprising the cyclin-binding motif and the Cdk inhibitory motif were obtained as a purified GST fusion protein
expressed in Escherichia coli (p21N-GST, gift of N. Furuno, N. den Elzen, and J. Pines, Wellcome/CRC Institute, University of
Cambridge, Cambridge, United Kingdom (Ref. 36)). As control, purified
GST was also used (gift of J. Pines). These compounds were added to the
reactions at the final concentrations indicated in the figures, and
control reactions contained equivalent volumes of the respective
solvents only.
Reactions containing protein phosphatase 
(New England Biolabs) were
supplemented with 20 µM MnCl2. Preincubations
in the absence of template DNA to allow dephosphorylation of
phosphoproteins were performed at 30 °C for 60 min. Single-stranded
M13mp18 DNA was then added to start complementary DNA strand synthesis
and minichromosomes assembly. Reactions were transferred to 37 °C for 120 min.
Nucleosome assembly in the absence of DNA synthesis was performed on
double-stranded M13mp18 DNA in the presence of cytosolic extract, a
buffered mix of ribo- and deoxyribonucleoside triphosphates, an
energy-regenerating system, and purified core histones as described (35). All reactions were mixed on ice and started by transferring to
37 °C. Standard reaction time was 120 min.
Processing of the Reaction Products--
Replication reactions
were stopped by the addition of 50 µl of 2× stop mix (2% sarcosyl,
0.2% SDS, 20 mM EDTA) and extracted in phenol-chloroform.
DNA was ethanol-precipitated, dissolved in TE buffer (10 mM
Tris-Cl, 1 mM EDTA, pH 8), and loaded onto 0.75% agarose
gels. Gel electrophoresis was performed at room temperature in 0.5×
TBE buffer (45 mM Tris borate, 0.5 mM EDTA, pH
8.4) at 3-4 V/cm. Two-dimensional gel electrophoresis (37) was
performed with 0.45-0.55 µM chloroquine in the second
dimension as detailed previously (9). DNA was visualized either by
autoradiography of the dried gel or by ethidium bromide staining after
RNase A digestion, as indicated in the figure legends.
Micrococcal nuclease (MNase) digestions were performed by
adjusting assembly reactions after 120 min to 3 mM
CaCl2 on ice. MNase (Roche) was added at the specified
amounts, and the reactions were transferred to 37 °C for 15 min.
Reactions were stopped by addition of 12.5 µl of 5× stop buffer
(2.5% sarcosyl, 100 mM EDTA), and the amount of
nuclease-resistant DNA was quantitated by trichloroacetic acid
precipitation and scintillation counting. For product analysis, nuclease-resistant DNA was purified from the stopped reactions and
analyzed on 1.5% agarose gels.
Protein Kinase Assays--
Recombinant cyclin/Cdk complexes
purified from Sf9 cells infected with recombinant baculovirus
expression vectors were gifts of M. Jackman, Dawn Coverley, and J. Pines (Wellcome/CRC Institute, University of Cambridge, Cambridge,
United Kingdom (Ref. 38)) and E. Laue and W. Zhang (Department of
Biochemistry, University of Cambridge). For in vitro
phosphorylation of the p60 subunit of CAF-1, recombinant p60 protein
was purified from E. coli C41 (BL21) (25). Purified p60 was
adjusted to kinase buffer (50 mM Tris, 1 mM
dithiothreitol, 5 mM ATP, 150 mM NaCl, and 10 mM MgCl2) using PD-10 column chromatography
(Amersham Pharmacia Biotech). Phosphorylation reactions were performed
in kinase buffer containing purified p60 (4 µg of protein), 0.1 µg
of protein of the recombinant cyclin/Cdk complexes, and 10 µCi of
[
-32P]ATP (Amersham Pharmacia Biotech) for 60 min at
30 °C. Samples were stopped and electrophoresed on a 12%
denaturating polyacrylamide gel, and proteins were stained by Coomassie
Blue. Phosphorylated proteins were detected by autoradiography of
the dried gel. Analysis of p60 phosphorylation by immunoblotting using
antibody pAb1 was performed as detailed previously (25).
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RESULTS |
Inhibition of Cyclin-dependent Protein Kinase Activity
Partially Inhibits Replication-dependent Nucleosome
Assembly in Human Cell Extracts--
Single-stranded circular DNA is
converted into double-stranded DNA upon incubation in cytosolic extract
from human cells, supplemented with ribo- and deoxyribonucleoside
triphosphates, radioactive dATP as a tracer, and an ATP-regenerating
system (9). Reaction products contain covalently closed relaxed
circular topoisomers, nicked circular form II DNA, and linear form III
DNA (Fig. 1A, lane
1). Addition of human CAF-1 triggers nucleosome assembly during complementary DNA strand synthesis (9), resulting in the
supercoiling of the covalently closed topoisomers into form I DNA (Fig.
1A, lane 2). Using this experimental
system, we first analyzed the influence of the Cdk inhibitor
roscovitine on nucleosome assembly by CAF-1 during complementary DNA
strand synthesis. Roscovitine specifically inhibits purified Cdks 1, 2, and 5 complexed to their cognate cyclin partners in vitro by
competing with ATP in a linear fashion at its binding site on the
kinases (39, 40). Addition of roscovitine up to a concentration of 0.5 mM reduced the amount of highly supercoiled form I DNA in a
dose-dependent manner (Fig. 1A, lanes
3 and 4). In contrast, topoisomers with lower
superhelical density were still present in these reactions, suggesting
that nucleosome assembly is less efficient in the presence of
roscovitine. With addition of roscovitine at a concentration of
1 mM or higher, DNA synthesis was also slightly inhibited
(Fig. 1A, lane 5 and data not shown); these
higher concentrations were therefore not used in the following
experiments.
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Fig. 1.
Cyclin E/Cdk2 and cyclin A/Cdk2 protein
kinase activity is required for efficient nucleosome assembly by CAF-1
during DNA synthesis. A, titration of the Cdk inhibitor
roscovitine. Complementary DNA strand synthesis reactions were
performed in the absence (left) and presence of CAF-1
(underlined). Reactions contained the indicated
concentrations of roscovitine. Reaction products were analyzed by
one-dimensional agarose gel electrophoresis and autoradiography.
Positions of DNA forms I (supercoiled), II (open circular), and III
(linear) are indicated. B, analysis of nucleosome assembly
by two-dimensional agarose gel electrophoresis. Reactions were
performed in the absence of CAF-1 (reaction 1), in the
presence of CAF-1 and 0.5 mM roscovitine (reaction
2), and in the presence of CAF-1 without roscovitine
(reaction 3). Purified DNA reaction products reactions were
applied to three slots along the diagonal of one two-dimensional gel.
Electrophoresis was run from top to bottom in the first dimension and
from left to right in the second dimension in the presence of 0.45 µM chloroquine. Reaction products were detected by
autoradiography. Positions of DNA forms II and III, and the median of
the topoisomer distributions (dots) are indicated for each
reaction (see Ref. 9 for further reference). C, quantitation
of the average number of nucleosomes assembled in these reactions.
Reactions 1-R3 of panel B
were repeated four to eight times, and reaction products were analyzed
on individual two-dimensional gels. The average changes in linking
number ( LK) of the median of the topoisomer
distributions, in comparison to the most relaxed topoisomer in the
populations, are presented as a histogram. One negative
LK corresponds to one nucleosome assembled during the
reaction. D, recombinant cyclin/Cdk2 complexes negate the
inhibition of CAF-1 activity by roscovitine. Reactions were performed
in the presence of CAF-1 and 0.5 mM roscovitine
(reactions 1-3), and supplemented with 1 µg of
recombinant cyclin A/Cdk2 (reaction 2) or cyclin E/Cdk2
(reaction 3). A control reaction was in the presence of
CAF-1 without roscovitine (reaction 4). Products were
analyzed by two-dimensional gel electrophoreses as in panel
B.
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We next determined the extent of inhibition of nucleosome assembly by
quantitating the number of supercoils formed in the presence of 0.5 mM roscovitine by two-dimensional agarose gel electrophoresis using chloroquine in the second dimension (9, 37).
After deproteinization of the reaction products, formation of one
negative supercoil, or a reduction of the linking number by one unit
(LK = 
1), corresponds to the presence of one
nucleosome originally assembled on the circular double-stranded DNA
molecule (41). The reference standard for this quantitation is the
relaxed topoisomer, which migrates most slowly during the first
dimension (LK = 0), due to the absence of
nucleosomes prior to deproteinization (9). The LK of
the median of a topoisomer distribution observed on a two-dimensional
gel therefore corresponds to the average number of nucleosomes formed
during an assembly reaction. Using this approach, we observed in the
absence of CAF-1 only a background assembly of a few nucleosomes during
complementary DNA strand synthesis (Fig. 1B, reaction
1; cf. Ref. 9). Addition of CAF-1 led to the assembly
of the maximal number of 38-40 nucleosomes (Fig. 1B,
reaction 3; cf. Ref. 9). Addition of
0.5 mM roscovitine significantly reduced the average number
of nucleosomes assembled (Fig. 1B, reaction
2). We quantitated the average LK of these reactions and observed that 0.5 mM roscovitine inhibits
nucleosome assembly by an average of about 50% (Fig. 1C).
We obtained similar results with the structurally related Cdk inhibitor
olomoucine (data not shown). These data suggest a requirement of a
cyclin-dependent kinase activity for efficient nucleosome
assembly by CAF-1 during DNA synthesis.
To directly test an involvement of Cdks we added recombinant human
cyclin/Cdk complexes to reactions containing both CAF-1 and roscovitine
and asked whether cyclin/Cdk complexes could negate the inhibition of
CAF-1 activity by roscovitine. Clearly, addition of cyclin A/Cdk2 or
cyclin E/Cdk2 fully negated the inhibition of
CAF-1-dependent nucleosome assembly by roscovitine (Fig.
1D). In control experiments, cyclin B1/Cdk1 or cyclin
D2/Cdk6 did not negate this inhibition (data not shown). These results
strongly suggest that cyclin A/Cdk2 and/or E/Cdk2 activity is required for nucleosome activity by CAF-1.
We sought to confirm independently a requirement of cyclin/Cdk2 for
CAF-1 activity during DNA synthesis by using a protein comprising the
N-terminal 90 amino acids from human p21 protein fused to GST
(p21N-GST; Ref. 36). The N terminus of p21 binds to and inhibits the
kinase activity of cyclin A/Cdk2, cyclin E/Cdk2, and cyclin D/Cdk4, but
not cyclin B1/Cdk1 (42-45). In contrast, the C terminus binds to PCNA
and inhibits DNA replication by an independent mechanism (42, 44,
46-48). Addition of purified p21N-GST protein clearly inhibited
nucleosome assembly by CAF-1 during complementary DNA strand synthesis
by 50% (Fig. 2A), whereas the
GST control protein did not inhibit nucleosome assembly (Fig. 2B).
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Fig. 2.
The Cdk inhibitor p21N inhibits nucleosome
assembly by CAF-1 during complementary DNA strand synthesis.
A, inhibition of CAF-1 activity by a p21N-GST fusion
protein. Reactions were performed in the absence of CAF-1
(reaction 1), in the presence of CAF-1 and 8 µM p21N (reaction 2), and in the presence of
CAF-1 without p21N (reaction 3). DNA reaction products were
analyzed by two-dimensional gel electrophoresis as detailed in the
legend to Fig. 1B, and the positions of topoisomers with 0, 10, 20, 30, or 40 negative supercoils are indicated along the
respective arcs. B, control reactions with GST protein.
Reactions were performed and analyzed as in panel
A, but p21N-GST was replaced by 8 µM GST in
reaction 2. For a better display of highly supercoiled DNA
topoisomers, an increased concentration of 0.55 µM
chloroquine was used in panel B.
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Taken together, these inhibitor and add-back experiments demonstrate
that the protein kinase activity of cyclin/Cdk2 complexes is required
for efficient nucleosome assembly by CAF-1 during complementary DNA
strand synthesis. However, about half of the nucleosome assembly
activity in this system appears to be resistant to the protein kinase
inhibitors and therefore not to depend on protein kinase activity. We
asked next whether inhibiting the reverse reaction, namely protein
dephosphorylation also influences nucleosome assembly during DNA synthesis.
Inhibition of Serine/Threonine Protein Phosphatase Activity
Partially Inhibits Replication-dependent Nucleosome
Assembly in Human Cell Extracts--
Addition of increasing amounts of
sodium vanadate neither inhibited complementary DNA strand synthesis
nor nucleosome assembly (Fig.
3A), indicating that protein
dephosphorylation at tyrosine residues is not essential in this system.
In contrast, addition of the inhibitor of the serine/threonine protein
phosphatase types 1 (PP1) and 2A (PP2A), okadaic acid, reduced the
amount of superhelical form I DNA with a compensating gain of relaxed
topoisomers (Fig. 3B). The number of nucleosomes assembled
by CAF-1 during complementary DNA strand synthesis in the presence of
okadaic acid was determined by two-dimensional gel electrophoresis
(Fig. 3C). Addition of 0.5 µM okadaic acid
reduced the average number of nucleosomes assembled by CAF-1 to about
60% (Fig. 3D). To discriminate between PP1 and PP2A, we
added increasing amounts of the PP1-specific inhibitor, I-2 (34, 49,
50). I-2 clearly inhibited the supercoiling of double-stranded reaction
products (Fig. 3B), suggesting that PP1 activity, rather
than PP2A activity, is required for efficient nucleosome assembly by
CAF-1 during DNA synthesis. In support of this conclusion, addition of
recombinant PP1 to assembly reactions negated the inhibition by okadaic
acid (Fig. 1D).
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Fig. 3.
Protein phosphatase type 1 (PP1) activity is required for efficient nucleosome
assembly by CAF-1. A, titration of the protein tyrosine
phosphatase inhibitor sodium vanadate. Complementary DNA strand
synthesis reactions were performed in the absence (left) and
presence of CAF-1 (underlined). Reactions contained the
indicated concentrations of sodium vanadate. Reaction products were
analyzed as detailed for Fig. 1A. B, titration of
the protein serine/threonine phosphatase inhibitor okadaic acid and the
PP1-specific inhibitor, I-2. Reactions contained CAF-1 and the
indicated concentrations of the inhibitors. Reaction products are
analyzed as in panel A. C, analysis of
nucleosome assembly by two-dimensional gel electrophoresis. Reactions
were performed in the absence of CAF-1 (reaction 1), in the
presence of CAF-1 and 0.5 µM okadaic acid (OA,
reaction 2), and in the presence of CAF-1 without okadaic
acid (reaction 3), and were analyzed as detailed for Fig.
1B. D, quantitation of the inhibition by 0.5 µM okadaic acid of nucleosome assembly by CAF-1 and
negation of this inhibition by recombinant human PP1. Reactions
1-R3 of panel C and a reaction containing
CAF-1, 0.5 µM okadaic acid, and recombinant human PP1
were analyzed on two-dimensional gels four to eight times, and data are
presented as detailed for Fig. 1C.
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Nucleosome Assembly by CAF-1 during Semiconservative DNA
Replication also Depends on Cyclin-dependent Kinase and
Protein Phosphatase Activities--
So far, CAF-1 activity was
analyzed during complementary DNA strand synthesis using
single-stranded DNA templates. To assess whether the dependence of
CAF-1 activity on cyclin/Cdk and PP1 activities also exists for
nucleosome assembly by CAF-1 during semiconservative DNA replication of
double-stranded DNA, we performed similar experiments using the SV40
DNA replication system (Fig. 4).
Replication depends on cytosolic extract, double-stranded DNA
containing the SV40 origin and the addition of the initiator protein
and DNA helicase T antigen (8). However, the interaction of T antigen
with cytosolic DNA polymerase 
/primase also depends on reversible
serine/threonine phosphorylation (51, 52). We therefore tested first
the influence of roscovitine and okadaic acid on SV40 DNA replication
(Fig. 4A). Both compounds inhibited DNA replication
strongly, as expected, but in the presence of excess T antigen, some
covalently closed circular mature DNA replication products were still
formed in the presence of 0.5 mM roscovtine or 1 µM okadaic acid (Fig. 4A). Interestingly,
formation of replication intermediates was less sensitive to these
compounds (Fig. 4A). In the presence of CAF-1, efficient
nucleosome assembly was strongly inhibited by roscovitine and by
okadaic acid (Fig. 4B), demonstrating that CAF-1 activity
during semiconservative replication of double-stranded DNA also depends
on cyclin/Cdk and protein phosphatase activity.
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Fig. 4.
Nucleosome assembly by CAF-1 during
semiconservative replication of double-stranded DNA containing the SV40
origin also depends on cyclin-Cdk and PP1 activity. A,
sensitivity of SV40 T antigen and origin-dependent DNA
replication to roscovitine and okadaic acid. Double-stranded M13O1 DNA
containing the SV40 control region (35) was replicated in the presence
of the indicated amounts of roscovitine (ros) and okadaic
acid (OA). Replication products were analyzed by gel
electrophoresis and autoradiography. Positions of DNA forms II and III
and of intermediates of semiconservative and bidirectional replication
containing  and  structures (RI) are indicated.
B, nucleosome assembly by CAF-1 during semiconservative
replication of double-stranded DNA is inhibited by roscovitine and
okadaic acid. DNA replication reactions were performed in the absence
of CAF-1 (reaction 1), in the presence of CAF-1 and 0.5 mM roscovitine (reaction 2) or 1 µM okadic acid (reaction 3), and in the
presence of CAF-1 without inhibitors (reaction 4). Purified
DNA replication product reactions were analyzed by two-dimensional gel
electrophoresis and autoradiography as detailed for Fig. 1B.
The gel was overexposed to reveal the topoisomers of reactions 2 and
3.
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Taken together, these results suggest that protein phosphorylation by S
phase-specific cyclin/Cdk2 complexes and its reverse reaction, protein
dephosphorylation by PP1, are both required for an efficient nucleosome
assembly by CAF-1 during DNA replication. However, some nucleosome
assembly activity appears to be resistant to either of the two kinds of
inhibitors. It is thus possible that both types of partial inhibition
act independently of each other, or act in an additive way, or cancel
each other out. Therefore, we analyzed the influence of both inhibitors
together on nucleosome assembly during DNA synthesis.
Inhibition of Both Protein Kinase and Phosphatase Activity Prevents
Nucleosome Assembly by CAF-1--
Addition of both 0.5 mM
roscovitine and 0.5 µM okadaic acid to DNA
synthesis-dependent nucleosome assembly reactions resulted predominantly in the formation of linear form III and open circular form II double-stranded DNA synthesis products (data not shown). These
unligated DNA synthesis products thus do not allow analysis of
nucleosome assembly by the supercoiling assay. Therefore, we analyzed
nucleosome assembly in the presence and absence of these inhibitors by
MNase digestions of the double-stranded DNA reaction products (Fig.
5).
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Fig. 5.
Nucleosome assembly during DNA synthesis is
inhibited by roscovitine and okadaic acid. A,
calibration curves for degradation of chromatin by micrococcal nuclease
(MNase). Complementary DNA strand synthesis reactions were
supplemented with either saturating amounts of CAF-1 (6 µg; 100%
CAF-1, squares), semisaturating amounts of CAF-1 (3 µg;
50% CAF-1, circles), or no CAF-1 (0% CAF-1,
triangles). Reaction products were digested with 0, 1, or 5 units of MNase at 37 °C for 15 min. Nuclease-resistant DNA was
precipitated with trichloroacetic acid and quantitated by scintillation
counting. B, quantitation of chromatin assembled in the
presence of roscovitine and okadaic acid. Assembly reactions in the
presence of saturating amounts of CAF-1 were supplemented with
Me2SO as control (squares), 0.5 mM
roscovitine (circles), 0.5 µM okadaic acid
(open triangles), and both roscovitine and
okadaic acid (open diamonds). Reaction products
were digested and quantitated as described in panel
A. Mean values of three to five independent experiments are
shown for each panel. C, analysis of nuclease-resistant DNA
by agarose gel electrophoresis. Assembly reactions were performed as in
panel B, and reaction products were digested with
0, 0.2, 1, and 5 units of MNase at 37 °C for 15 min.
Nuclease-resistant DNA was purified by ethanol precipitation, separated
on a 1.5% agarose gel, and visualized by autoradiography. Positions of
uncut DNA forms I, II, and III and of nuclease-resistant DNA fragments
of mono-, di-, tri-, and tetranucleosomal length are indicated on the
left, top, and bottom,
respectively.
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CAF-1 assembles nucleosomes during complementary DNA strand synthesis
in human cell extracts in a stoichiometric manner (9). As reference, we
first calibrated the resistance of chromatin assembled to various
nucleosomal densities to a digestion with MNase (Fig. 5A).
Minichromosomes assembled during complementary DNA strand synthesis in
the presence of saturating amounts of CAF-1 were 76% resistant to a 15 min digestion with 5 units of MNase (Fig. 5A,
squares), compared with an efficient degradation of reaction
products in the absence of CAF-1 to 40% (Fig. 5A, triangles). Reaction products assembled in the presence of
halfmaximal amounts of CAF-1 contain half the complement of nucleosomes
(9), and they are degraded by MNase to an intermediate value of 55% (Fig. 5A, circles).
Minichromosomes assembled in the presence of either 0.5 mM
roscovitine or 0.5 µM okadaic acid were resistant at
intermediate levels of 62% and 69% to degradation with MNase (Fig.
5B, circles and triangles,
respectively). These data suggest that only about half the maximal
number of nucleosomes are assembled in the presence of either 0.5 mM roscovitine or 0.5 µM okadaic acid, as
compared with the control in the absence of inhibitors (Fig.
5B, squares). These observations are entirely
consistent with, and independently confirm the supercoiling data of
Figs. 1 and 3.
Reaction products assembled in the presence of both inhibitors were
efficiently degraded by MNase to 49% (Fig. 5B,
diamonds), suggesting that only very few nucleosomes are
assembled during this reaction. These data establish that the
inhibitory effects of cyclin/Cdk and protein phosphatase inhibitors act
in an additive way on chromatin assembly during DNA synthesis.
These quantitative data were supported by the analysis of the
nuclease-resistant DNA products by agarose gel electrophoresis (Fig.
5C). Reaction products assembled in the absence of either inhibitor showed nuclease-resistant DNA fragments of mono- and spaced
oligonucleosomal length (Fig. 5C, control).
Addition of either roscovitine or okadaic acid resulted in an increased
nuclease sensitivity of the reaction products, apparent in the shorter average fragment length in the presence of 0.2 unit of MNase and the
presence of smaller amounts of predominantly mononucleosomal fragments
at higher concentrations of MNase (Fig. 5C). In the presence
of both inhibitors, roscovitine and okadaic acid, reaction products
were efficiently degraded, and only trace amounts of mononucleosomal
DNA fragments were observed.
Taken together, these results show that nucleosome assembly by CAF-1
during complementary DNA strand synthesis is strongly inhibited, if not
prevented, when both protein kinase and phosphatase activities are
inhibited. This observation could be explained either by a general
requirement of these activities for an assembly of nucleosomes in human
cell extracts or by a specific requirement for the DNA
synthesis-dependent nucleosome assembly pathway mediated by
CAF-1. To distinguish between these possibilities, we asked whether
replication-independent nucleosome assembly on double-stranded DNA in
human cytosolic extracts is also inhibited by these inhibitors.
Nucleosome Assembly in the Absence of DNA Synthesis Is Not
Inhibited by Roscovitine or Okadaic Acid--
Nucleosomes can be
efficiently assembled in human cytosolic extracts on double-stranded
DNA in the absence of DNA replication and CAF-1 by a different pathway,
which relies on purified core histones and a cytosolic assembly factor
(35, 53). Addition of core histones purified from human interphase
nuclei into reactions containing circular double-stranded DNA
substrates and cytosolic extract induced formation of supercoiled form
I DNA in a dose-dependent manner (Fig.
6A; cf. Refs. 35
and 53). Importantly, addition of either roscovitine or okadaic acid,
or a combination of both, did not inhibit this nucleosome assembly
pathway at any concentrations of core histones (Fig. 6A). We
furthermore analyzed possible subtle changes in the efficiency of this
nucleosome assembly by gel electrophoresis in the presence of
chloroquine (Fig. 6B). As control, addition of either or
both compounds did not significantly affect the relaxation of
supercoiled DNA by cytosolic topoisomerase (Fig. 6B,
core histones). Upon addition of a 1:1 mass
ratio of core histones to the DNA, nucleosome assembly as measured by
constrained DNA supercoiling was also not affected by either, or both,
of roscovitine and okadaic acid (Fig. 6B, +core
histones). These results demonstrate that protein kinase and
phosphatase activities are not required for nucleosome assembly in
human cell extracts per se, but they are specifically
required for the assembly pathway mediated by CAF-1 during DNA
synthesis.
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Fig. 6.
Nucleosome assembly on double-stranded DNA in
the absence of DNA synthesis is not dependent on protein
serine/threonine kinase or phosphatase activities. A,
titration of core histones. 200 µg of double stranded circular
M13mp18 DNA was incubated in the presence of 0, 100, 200, and 400 µg
of purified core histones in the absence of inhibitors
(lanes 2-5) and in the presence of 0.5 mM roscovitine (lanes 6-8), or 1 µM okadaic acid (lanes 9-11), or
both inhibitors together (lanes 12-14). Markers
were supercoiled form I DNA (lane 1) and DNA
relaxed by endogenous topoisomerases in the extract in the absence of
core histones (lane 2). Reaction products were
deproteinized and analyzed on a native 0.75% agarose gel. DNA was
visualized by staining with ethidium bromide, and the positions of DNA
forms I and II are indicated. B, analysis of nucleosome
assembly by one-dimensional gel electrophoresis in the presence of 0.55 µM chloroquine. Nucleosome assembly reactions containing
200 µg of DNA and either 0 µg (left) or 200 µg
(right) of core hisones were supplemented with reaction
buffer (), Me2SO (D), 0.5 mM
roscovitine (r), 1 µM okadaic acid
(o), or both inhibitors (r/o). Marker was
supercoiled form I DNA (C). Reaction products were analyzed
on a 0.75% agarose gel containing 0.55 µM chloroquine,
and DNA was visualized by staining with ethidium bromide. The positions
of DNA forms II and III and the populations of covalently closed
relaxed (Irel) and form I DNA topoisomers
(I) are indicated.
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In intact human cells, previous correlative studies described
phosphorylation changes of the p60 subunit upon recruitment of CAF-1 to
intranuclear sites of DNA replication and repair (17, 25). We therefore
investigated in human cell extracts whether CAF-1 is a target for
reversible protein phosphorylation during complementary DNA strand
synthesis and nucleosome assembly.
CAF-1 Is Targeted by Phosphorylation and Dephosphorylation during
DNA Synthesis-dependent Nucleosome Assembly--
The
phosphorylation state of p60 was analyzed by Western blotting (Fig.
7A), using antibody pAb1,
which detects the phosphorylated isoforms of p60 (25). The p60 subunit
of CAF-1 prepared from interphase nuclei consists of two distinct
forms, a dephosphorylated form (c) and an intermediately
phosphorylated form (b), whereas in mitosis, p60 appears as
hyperphosphorylated form (a) (Ref. 25, and see Fig.
7A).
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Fig. 7.
Phosphorylation of the p60 subunit of human
CAF-1 during complementary DNA strand synthesis is changed by the
activities of cyclin-dependent kinases and protein
phosphatases 1 and 2A. A, CAF-1 isolated from
interphase nuclei was added to complementary DNA strand synthesis
reactions in the presence of the indicated concentrations of
inhibitors. Proteins of these reactions were separated on a 8%
SDS-polyacrylamide gel and analyzed by Western blotting using
polyclonal antibody pAb1 (25), which detects the different
phosphorylated isoforms of the p60 subunit of CAF-1.
Hyperphosphorylated form (a), intermediate form
(b) and hypophosphorylated form (c) are indicated
for each panel. B, direct phosphorylation of the p60 subunit
of CAF-1 by S phase-specific and by mitotic cyclin/Cdks in
vitro. Protein kinase assays were performed with 4 µg of
purified recombinant p60 and 0.1 µg of the indicated cyclin/Cdk
complexes. The protein kinase activity of these complexes was confirmed
on histone H1 and pRb as templates in parallel incubations (data not
shown). Protein samples were separated on denaturing 12%
polyacrylamide gels, and proteins were stained with Coomassie Blue.
Protein phosphorylation was detected by autoradiography. The position
of phosphorylated p60 (corresponding to form b, data not
shown) is indicated on the left. C,
hyperphosphorylation of p60 depends on cyclin B1/Cdk1 and additional
cytosolic factors. CAF-1 was added to the indicated amounts of
recombinant cyclin/Cdk complexes in the presence of cytosolic extract
and single-stranded DNA. After 120 min, phosphorylation of p60 was
analyzed by Western blotting.
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Addition of increasing amounts of roscovitine to nucleosome assembly
reactions during complementary DNA strand synthesis resulted in a
partial, dose-dependent dephosphorylation of the p60
subunit (Fig. 7A). Conversely, addition of increasing
amounts of okadaic acid resulted in a partial,
dose-dependent hyperphosphorylation of the p60 subunit
(Fig. 7A). Adding both inhibitors together did not result in
either dephosphorylation or hyperphosphorylation of p60, but resulted
predominantly in the intermediately phosphorylated form (b)
of p60 (Fig. 7A). In conjunction with the results of the
previous sections, these data show that CAF-1 is targeted by ongoing
reversible protein phosphorylation, which is required for the
nucleosome assembly activity of CAF-1 during complementary DNA strand
synthesis. We asked next whether p60 can be directly phosphorylated by
purified cyclin-dependent kinases.
Recombinant p60 was purified from transformed bacteria (25) and used as
in vitro substrate for phosphorylation by recombinant human
cyclin/Cdk complexes, prepared from baculovirus-infected insect cells
(38). Analysis of the amino acid sequence of p60 (13) revealed two Cdk
phosphorylation motifs fitting the consensus sequence
(T/S)PX(K/R) and several (T/S)PX motifs in the C
terminus of p60 (data not shown). The G1/S phase-specific
cyclin E/Cdk2 and cyclin A/Cdk2 complexes mediated direct
phosphorylation of p60 (Fig. 7B). Furthermore, mitotic
cyclin B1/Cdk1 also phosphorylated p60 (Fig. 7B). In
contrast, cyclin D1/Cdk4 as a representative of the cyclin D-Cdk4/6
protein kinase family did not phosphorylate p60 (Fig. 7B).
We obtained similar results when CAF-1 prepared from human cell nuclei
was used as substrate (data not shown). Therefore, S and M
phase-specific cyclin/Cdk complexes can directly phosphorylate p60
in vitro.
In this assay, phosphorylation of recombinant p60 by cyclin/Cdks did
not result in formation of the hyperphosphorylated form (a)
of p60 (Fig. 7B), which is observed when nucleosome assembly in human cell extracts is inhibited by okadaic acid (Fig.
7A), or when CAF-1 is prepared from mitotic cells (Ref. 25;
cf. Fig. 7A). We therefore asked whether this
mobility shift is dependent on additional factors present in the
cytosolic extract. Indeed, addition of recombinant cyclin B1/Cdk1, but
not of cyclin A/Cdk2, to DNA synthesis reactions resulted in the
electrophoretic mobility shift characteristic for hyperphosphorylated
p60 (Fig. 7C), suggesting that cyclin B1/Cdk1 is required in
conjunction with other factors or modifications to induce
hyperphosphorylation of p60. As mitotic CAF-1, containing a
hyperphosphorylated p60 subunit, is inactive in supporting nucleosome
assembly in vitro (25), we finally asked whether inactive
hyperphosphorylated mitotic CAF-1 can be functionally activated in
human extracts by dephosphorylation.
Reconstitution of Nucleosome Assembly Activity of
Hyperphosphorylated Mitotic CAF-1 by Dephosphorylation--
CAF-1 was
prepared from mitotic chromatin and used in nucleosome assembly
reactions during complementary DNA strand synthesis in human
G1 phase cytosolic extracts. As shown before (25), p60 of
mitotic CAF-1 is hyperphosphorylated (Fig.
8A) and does not support
efficient nucleosome assembly in vitro (Fig. 8B,
lane C). Treatment with increasing amounts of phosphatase led to a dephosphorylation of the p60 subunit of CAF-1 in
the cytosolic extract (Fig. 8A). Even in the absence of phosphatase, a limited dephosphorylation of p60 occurred by endogenous
phosphatase activity in the extract, which is activated by the addition
of 20 µM MnCl2 (Fig. 8A,
reaction 1). We then added single-stranded template DNA to
these reactions to start complementary DNA strand synthesis and
nucleosome assembly. The extent of p60 phosphorylation was not
significantly altered during this subsequent incubation (Fig. 8A). Analysis of DNA reaction products demonstrated that
dephosphorylation of CAF-1 during preincubation resulted in the
formation of supercoiled form I DNA products (Fig. 8B).
These data demonstrate that dephosphorylation of inactive
hyperphosphorylated CAF-1 causes the activation of its nucleosome
assembly activity during DNA synthesis, thus adding further evidence
for the requirement of reversible phosphorylation for nucleosome
assembly by CAF-1 during DNA synthesis.
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Fig. 8.
Reconstitution of nucleosome assembly
activity by dephosphorylation of inactive hyperphosphorylated CAF-1
from mitotic HeLa cells. A, changes of the
phosphorylation of the p60 subunit in vitro. CAF-1 isolated
from mitotic chromatin was preincubated for 60 min in the presence of
cytosolic extract from G1 phase cells, 20 µM
MnCl2, and 0 (, reaction 1), 400 (+,
reaction 2), and 800 units of phosphatase (+++,
reaction 3), but in the absence of single-stranded DNA.
Complementary DNA strand synthesis was started by adding DNA and
incubated for another 120 min. Protein samples were taken after
preincubation and after the entire reaction time. The p60 subunit of
CAF-1 was detected by Western blotting using polyclonal antibody pAb1.
As control, samples of CAF-1 prepared from interphase nuclei and from
mitotic chromatin are shown on the left. B,
nucleosome assembly during complementary DNA strand synthesis. CAF-1
isolated from mitotic chromatin was preincubated for 60 min in the
presence of cytosolic extract from G1 phase cells, 20 µM MnCl2 and 0 (lane
1), 400 (lane 2), and 800 units of phosphatase (lane 3). Complementary DNA strand
synthesis was started by adding single-stranded DNA and incubated for
another 120 min. As control (lane C),
complementary DNA strand synthesis was performed using inactive
hyperphosphorylated mitotic CAF-1 and G1 phase cytosolic
extract, but without MnCl2 and phosphatase (see Ref.
25). DNA synthesis products were investigated by agarose gel
electrophoresis and autoradiography.
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DISCUSSION |
The major conclusion from this work is that nucleosome assembly by
CAF-1 during DNA synthesis in human cell extracts depends on reversible
protein phosphorylation involving G1/S phase-specific cyclin-dependent protein kinase 2 and type 1 protein
phosphatase. The p60 subunit of CAF-1 changes its phosphorylation state
during nucleosome assembly in human cytosolic cell extract and becomes dephosphorylated in the presence of Cdk inhibitors and
hyperphosphorylated when PP1 is inhibited. Purified p60 can also be
directly phosphorylated by purified cyclin/Cdk kinases in
vitro, suggesting that CAF-1 is a molecular target for reversible
phosphorylation. We cannot exclude an involvement of additional kinase
and phosphatase activities during reversible phosphorylation of CAF-1
in cell extracts during nucleosome assembly. However, a defined
experimental system that uses purified components for
replication-dependent nucleosome assembly by CAF-1 has not
been reported to date to identify all the essential molecules during
this reaction. Therefore, our results provide the first functional
evidence for a requirement of the main players of the cell cycle
machinery for CAF-1 activity during DNA synthesis.
Requirement of Reversible Protein Phosphorylation for Nucleosome
Assembly by CAF-1--
We studied the requirement of reversible
protein phosphorylation for nucleosome assembly by CAF-1 during DNA
synthesis in human cell extracts by using natural compounds, or their
synthetic derivatives, as inhibitors. Roscovitine and the related
compound olomoucine specifically inhibit purified Cdks 1, 2, and 5 complexed to their cognate cyclin partners in vitro by
competing with ATP in a linear fashion at its binding site on the
kinases (39, 40, 54). We found that a 0.5 mM concentration
of roscovitine inhibited CAF-1 activity, whereas increased
concentrations also inhibited DNA replication. In supplementation
experiments, we demonstrated that the addition of recombinant cyclin
A/Cdk2 and E/Cdk2 complexes negated the inhibitory effect of 0.5 mM roscovitine on CAF-1 activity. Addition of cyclin
B1/Cdk1 or D/Cdk6 did not. These experiments strongly suggest that the
kinase activity of Cdk2, when complexed to either cyclin A or E, is
required for nucleosome assembly by CAF-1.
This conclusion is supported by the sensitivity of CAF-1 activity to
inhibition by the amino terminus of the natural human Cdk inhibitor
p21. p21 directly binds to and significantly inhibits the kinase
activity of cyclin A/Cdk2, cyclin E/Cdk2 (and cyclin D/Cdk4), but not
cyclin B/Cdk1 (45). The amino terminus of p21 contains the binding
motifs for cyclins and Cdks (42-44). Microinjection experiments
demonstrated an inhibition by p21N of cyclin A/Cdk2 but not of cyclin
B1/Cdk1 in human cells at a concentration similar to the one used in
our in vitro studies (36). We deliberately used p21N rather
than the entire p21 protein in order to exclude inhibitory reactions
through interaction of the C terminus of p21 with PCNA (42, 48). PCNA
has independently been shown to be a factor involved in targeting CAF-1
to DNA templates undergoing replication (20) and repair (18).
Taken together, these experiments established that the G1/S
phase-specific cyclin/Cdk2 complexes, which are required for initiating DNA replication in isolated human cell nuclei (38, 55), are also
required for nucleosome assembly by CAF-1 during DNA replication in vitro.
Okadaic acid is a specific inhibitor of the protein phosphatases PP1
and PP2A, which have broad and overlapping substrate specificities,
including serine and threonine residues phosphorylated by Cdks (34, 50,
56). Inhibition of CAF-1 activity by okadaic acid functionally
demonstrates a role of either PP1 or PP2A for nucleosome assembly by
CAF-1 during DNA synthesis. The PP1-specific peptide inhibitor I-2,
which does not inhibit PP2A (50), also inhibited CAF-1 activity,
indicating that PP1, rather than PP2A, is the type of protein
phosphatase required for CAF-1 activity during DNA synthesis.
Furthermore, in supplementation experiments, the inhibition of CAF-1
activity by okadaic acid was entirely relieved by addition of the
purified recombinant catalytic subunit of human PP1. Taken together,
these experiments established a requirement of PP1 activity for
nucleosome assembly by CAF-1 during DNA replication in
vitro.
Changes of CAF-1 Phosphorylation during the Cell Cycle in
Vivo--
Our experiments established functional requirements of
cyclin/Cdk2 complexes and PP1 for nucleosome assembly by CAF-1 during DNA replication in human cell extracts. Furthermore, the
phosphorylation state of the p60 subunit changes accordingly in the
presence of the inhibitors used (Fig. 7A) and p60 can be
directly phosphorylated by purified cyclin/Cdk complexes (Fig.
7B). These experiments demonstrate that CAF-1 is a target
for ongoing reversible protein phosphorylation in the cell extracts
used for nucleosome assembly and DNA synthesis in vitro.
However, these data cannot rule out an indirect involvement of
additional kinases and phosphatases, which could also be required for
reversibly phosphorylating p60 during these assembly reactions in
vitro.
Roles of cyclin/Cdk complexes and PP1 for regulating CAF-1 activity
during chromosome dynamics in the cell cycle in vivo are supported by their respective intracellular localizations in human and
other mammalian somatic cells. During S phase, CAF-1 co-localizes with
intranuclear replication foci (24). Cyclins A, E, and Cdk2 are nuclear
proteins in S phase (57, 58), and cyclin A and Cdk2 have been shown to
co-localize with replication foci during S phase (59). These
observations support a physiological role of these proteins in
chromosome replication, and one further function of these cyclin/Cdk
complexes may be the phosphorylation of CAF-1.
The intracellular localization of PP1 during the cell cycle also
supports a physiological role in chromosome replication. The catalytic
subunit of PP1 is predominantly cytosolic in mammalian G1
phase cells and accumulates in nuclei during S and G2
phases (60). Analysis of the three different subtypes of PP1 catalytic subunit showed nuclear localization of all subtypes during interphase and a specific association of PP1
with chromatin (61), suggesting that this subtype is a likely candidate for regulating chromatin assembly during DNA replication in S phase nuclei. Therefore, cyclin
A/Cdk2, cyclin E/Cdk2, and PP1, which are required for efficient
nucleosome assembly by CAF-1 during DNA replication in vitro
(this paper), are at the nuclear location of DNA replication and
nucleosome assembly during S phase in vivo.
In mitosis, the p60 subunit of CAF-1 is hyperphosphorylated and CAF-1
prepared from mitotic cells does not support nucleosome assembly
in vitro (25). Mitotic cyclin B1/Cdk1 can hyperphosphorylate p60 in vitro, and its protein kinase activity is not
required for nucleosome assembly by CAF-1 (this paper). Cyclin B1
enters the nucleus at the beginning of mitosis and associates with
condensed chromosomes in prophase and metaphase (57), consistent with a
role in hyperphosphorylating p60, histone H1 and other mitotic substrates. A functional role for the cyclin
B1/Cdk1-dependent hyperphosphorylation of p60 in reversibly
inactivating nucleosome assembly activity of CAF-1 in mitosis was
supported by our observation that dephosphorylation of inactive mitotic
CAF-1 in vitro restores its nucleosome assembly activity
(Fig. 8). The p60 subunit of CAF-1 appears fully dephosphorylated in
the presence of excess phosphatase, and nucleosome assembly by
CAF-1 is activated. (Note that this experiment does not contradict our
inhibitor studies showing that an inhibition of Cdk activity results in
dephosphorylation of p60 and an inhibition of nucleosome assembly by
CAF-1. In the presence of excess of phosphatase the endogenous Cdk
activity in the extract is not inhibited, allowing reversible
phosphorylation to continue with the equilibrium of p60 phosphorylation
being shifted toward the dephosphorylated form.)
PP1 is bound to condensed chromosomes throughout mitosis (60, 61), and
its phosphatase activity is required for the metaphase to anaphase
transition and exit from mitosis (60), coincident with cyclin B1
destruction (57). In human cells, PP1 activity has also been found as
histone H1 phosphatase, which is required for chromatin decondensation
during exit from mitosis (62). During the M to G1 phase
transition, the hyperphosphorylation of p60 is also removed (25),
consistent with a possible role for PP1 in dephosphorylating p60 and
other substrates upon exit from mitosis.
Models for a Requirement of Reversible Phosphorylation for CAF-1
Activity--
We observed that inhibiting either directions of
reversible protein phosphorylation, namely phosphorylation by
cyclin/Cdk complexes and dephosphorylation by PP1 partially inhibits
nucleosome assembly by CAF-1 (Figs. 1-5). These apparently opposing
observations can be explained by different working models.
In a simplistic model, only phosphorylation by Cdk2 would be directly
required for nucleosome assembly by CAF-1. In this model, inhibition of
PP1 by okadaic acid would allow accumulation of hyperphosphorylated
CAF-1 generated by small amounts of cyclin B1/Cdk1 kinase activity
present in the extracts from asynchronous cells. Indeed,
hyperphosphorylated p60 is observed in the presence of okadaic acid in
the reaction (Fig. 7A), and CAF-1 containing hyperphosphorylated p60 does not promote efficient nucleosome assembly
(Figs. 3, 7A, and 8). These data are consistent with our
previous observation that CAF-1 containing hyperphosphorylated p60 is
inactive when isolated from mitotic cells (Fig. 8 and Ref. 25).
However, this model cannot easily explain the significant residual
CAF-1 activity in the presence of okadaic acid or roscovitine/p21N. It
is also difficult to accommodate into this working model the additive
inhibitory effect on CAF-1 activity when both protein phosphorylation
and dephosphorylation are inhibited at the same time (Fig. 5).
Instead, several mechanistic working models involving reaction cycles
could explain our observations (Fig. 9).
CAF-1 is a histone chaperone and assembles nucleosomes in a
non-cooperative and stoichiometric fashion during DNA synthesis in
human cell extracts (9, 13, 15, 17). All three subunits of CAF-1 are
histone-binding proteins (12, 13, 63). In the first working model,
reversible binding of histones to CAF-1 may depend on the
phosphorylation status of CAF-1. The binding of newly synthesized histones to CAF-1 forming CAC (Fig. 9, reaction 1) and the
release of histones onto double-stranded DNA to form a nucleosome (Fig. 9, reaction 3) may require a cycle of reversible
phosphorylation and dephosphorylation.
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Fig. 9.
Pathways of CAF-1-mediated nucleosome
assembly during DNA replication that may be regulated by reversible
protein phosphorylation. 1, association of CAF-1 with
newly synthesized histones forming the chromatin assembly complex CAC.
2, association of CAC with replicating DNA to form an
assembly intermediate, involving interaction of p150 with replication
fork-associated protein PCNA. 3, formation of a newly
assembled nucleosome from the assembly intermediate. 4,
detachment of CAF-1 from the intermediate. For more details, see
references (2, 5, 15). Reversible pathways during the CAF-1 reaction
cycle that could be regulated by reversible phosphorylation, involving
the p60 subunit, are the association with and the dissociation from
replicated DNA (reactions 2 and 4 along the vertical;
open arrows), or the association with and the
dissociation from histones (reactions 1 and 3 shown along the horizontal; solid
arrows).
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In the second working model, the reversible association of CAF-1 with
replicating DNA, involving interaction with replication fork associated
protein PCNA, could depend on reversible phosphorylation. CAF-1 is
present at replication foci in S phase nuclei (24), and it is recruited
to the replication fork involving an association of p150 with PCNA (18,
20). The association of CAC with the replicating DNA to form an
assembly intermediate (Fig. 9, reaction 2), and the release
of CAF-1 from this intermediate to allow formation of a mature new
nucleosome (Fig. 9, reaction 4) may require a cycle of
reversible phosphorylation and dephosphorylation.
These two working models involving reaction cycles would also allow
reconciliation of the partial nature of the inhibition of nucleosome
assembly by CAF-1 by inhibiting Cdk2 and PP1 activities. The starting
material of CAF-1 used for nucleosome assembly in this study consists
of a mixture of unphosphorylated and phosphorylated p60 (Fig. 7).
Conversion of one form of p60 into the other during nucleosome assembly
in vitro, while preventing the reverse reaction by
inhibitors, would therefore still be possible for about half of the p60
present in the reaction. This inhibitor-resistant conversion could be
responsible for the residual CAF-1 activity observed in the presence of
either inhibitor alone, but not in the presence of both inhibitors together.
We cannot currently discern whether dephosphorylated or phosphorylated
CAF-1 is at the start of any of those reversible phosphorylation cycles. However, during the cell division cycle in vivo,
phosphorylated CAF-1 is associated with chromatin during G1
phase and the occurrence of a dephosphorylated form of p60 is
characteristic for S phase (25). During repair of UV-induced DNA damage
in human G2 phase cell nuclei, the phosphorylated form of
p60 is found in a detergent-resistant chromatin fraction, whereas in
non-irradiated G2 phase cells, p60 is predominantly
dephosphorylated in this fraction (17). These results may suggest that
recruitment of CAF-1 to sites of DNA synthesis may first require
phosphorylation, and dissociation would then depend on dephosphorylation.
Future experiments using purified components with defined
phosphorylation status will be therefore required to discriminate between these scenarios on a molecular level. To date, no experimental system exists that mediates nucleosome assembly by CAF-1 during DNA
synthesis using purified components. However, the identification of two
novel factors reported here, namely cyclin/Cdk2 and protein phosphatase
type 1, which are required for efficient nucleosome assembly by CAF-1
during DNA synthesis in unfractionated human cell extracts will help
future attempts to establish such defined cell-free systems from
purified components.
and
TOP
ABSTRACT
INTRODUCTION
Present address: Dept. of Biology, University of Konstanz, D-78464
Konstanz, Germany.
