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INTRODUCTION |
Initiation of genome replication has attracted considerable
attention in recent years, because it is an essential and tightly regulated process within the eukaryotic cell cycle. Work with yeast
mutants has been and continues to be instrumental in the identification
and characterization of proteins involved in the initiation of
eukaryotic genome replication. An important step toward understanding
the events at replication start sites was the discovery of a complex of
six proteins, Orc1p-Orc6p, that specifically recognize the yeast
origins of replication (1). The origin recognition complex
(ORC)1 appears to be stably
bound to chromatin sites during all cell cycle phases in yeast (2-5)
and recruits early in the G1 cell cycle phase additional
initiation factors, including the Cdc6 protein and the Cdt1 protein (3,
6-8). Yeast Cdc6p appears to function as an ATP-dependent
loading factor for the six-membered Mcm group of initiation
proteins (Mcm2p-Mcm7p) (9, 10). The transition from G1 to S
phase is coordinated by the timely activation of protein kinases such
as the Cdc7/Dbf4 kinase and S phase-promoting cyclin-dependent kinases (see reviews in Refs. 11-13). It
is characterized by a dissociation of Cdc6p and the Cdc45p-mediated
assembly of DNA replication factors such as the single strand-specific
DNA-binding protein RPA and other proteins required for the
establishment of replication forks (11, 14-16). Possibly, Mcm proteins
execute their intrinsic DNA helicase activity (17) at or just prior to
this stage and unwind the DNA sequences to which they are bound.
A similar general scheme for the assembly of early initiation proteins
has been derived from biochemical studies with the Xenopus
system. In Xenopus cell-free egg extracts,
Xenopus Cdc6p (xCdc6p) can only bind to chromatin carrying
Orc proteins, and binding of xCdc6p was shown to be essential for the
subsequent loading of xMcm3p and other Mcm proteins (18-21). However,
the Xenopus ORCs undergo interesting transitions once the
prereplication complex has been assembled. Interactions of xOrc
proteins and xCdc6p with their chromatin binding sites become
destabilized and sensitive to high salt concentrations (22) and to the
activity of a cyclin A-dependent kinase (23). In fact, xOrc
proteins appear to be no longer required after completion of
prereplication complex formation (22-24), and xOrc protein cannot be
detected on mitotic chromatin (18).
Cell cycle-dependent variations in Orc proteins have also
been detected studying Orc1p in developing Drosophila
embryos. Drosophila Orc1p can only be detected in actively
proliferating cells as well as in cells arrested in the G1
phase, but not in cells arrested after completion of DNA replication
(25, 26).
Thus, even though the overall mechanism of prereplication complex
formation is probably conserved among eukaryotes, interesting differences appear to exist between yeast and metazoan ORCs as yeast
ORCs remain stably bound to origins during all cell cycle phases
whereas the interactions of Xenopus and
Drosophila ORCs with their cognate chromatin sites seem to
vary during the cell cycle. Recent evidence suggests that this
conclusion may not be restricted to the early developmental stages of
Xenopus and Drosophila systems but applies to
cultured mammalian cells as well (27). It has been shown that nuclei
prepared from hamster cells just released from a mitotic nocodazole
block can only be induced to replicate their genomes when incubated in
Xenopus egg extracts with sufficient quantities of xOrc1p.
Exogenous xOrc1p initiates replication at sites in the hamster genome
that are not normally used as replication origins. In contrast, hamster
cell nuclei prepared at later times after release from the nocodazole
block do not depend on exogenous xOrc1p, but have assembled their own functional ORCs with endogenous Orc1p and are able to activate genuine
hamster cell origins (27). These important findings suggest that
functional mammalian ORCs assemble each time a cell enters the
G1 phase.
However, we have previously shown that human Orc2p (hOrc2p) remains
bound to chromatin during the entire HeLa cell cycle (28), an
observation that appears to be in conflict with the possibility of a
cell cycle-dependent assembly of new ORCs. To better
understand this situation we now report on experiments that address
this issue and describe hOrc1p dissociation from its chromatin binding sites during S phase together with a fraction of hOrc2p that is complexed to hOrc1p. Another and possibly larger fraction of hOrc2p appears not to be complexed with hOrc1p and occurs in a different chromatin compartment. It is this fraction of hOrc2p that remains bound
to chromatin during the entire cell cycle.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human HeLa S3 cells were grown on plastic
dishes in Dulbecco's modified Eagle's medium plus 5% fetal calf
serum. Cells were synchronized by a double thymidine block at the
beginning of the S phase and released into thymidine-free medium (29).
S phase was determined by pulse-labeling with
[3H]thymidine and mitosis by determining mitotic indices
exactly as described before (see Fig. 1 in Ref. 28).
Cell Fractionation--
Cells were washed on plates with
phosphate-buffered saline and suspended in hypotonic buffer A (20 mM Hepes, 20 mM NaCl, 5 mM
MgCl2, 1 mM ATP, pH 7.5). After 15 min on ice,
the swollen cells were broken by Dounce homogenization and centrifuged
to separate the cytosolic supernatant from the nuclear pellet. Nuclei were resuspended in buffer A with 0.5% Nonidet P40 and kept on ice for
15 min to lyse the nuclear envelope. Centrifugation separated the free
nucleosolic proteins from the pelleted nuclei. The pellet was washed
with buffer B (20 mM Hepes, 0.5 mM
MgCl2, 1 mM ATP, 0.3 M sucrose, pH
7.5) plus NaCl in concentrations of 0.1-0.45 M (see below)
to release structure-bound proteins.
Chromatin Fractionation--
The isolation of S1,
S2, and P chromatin fractions was based on the
procedure described by Rose and Garrard (30). Nuclei were prepared as
described in "Cell Fractionation" and investigated by micrococcal
nuclease digestions performed in buffer B supplemented with 2 mM CaCl2 and 100 mM NaCl (final
concentration) under the conditions described in the "Results."
Digested chromatin was used for immunoblotting (see below) or
deproteinized (0.5% SDS, proteinase K: 200 µg/ml for 30 min at
37 °C) and investigated by agarose gel (0.8%) electrophoresis and
ethidium bromide staining.
Chromatin Preparation--
Chromatin was prepared under low
ionic strength conditions first described by Hancock (31) and detailed
in (28). DNA concentrations were determined with Hoechst 33528 by
fluorimetry (Hoefer Scientific Instruments, San Francisco).
Antibodies--
Complete cDNA sequences encoding human
proteins Orc1 and Orc2 have been described (32) and were kindly
provided by B. Stillman, Cold Spring Harbor. The cDNAs were
recloned in the expression vector pRSET (Invitrogen) and sequenced to
check the cloning results and the integrity of the coding regions.
His-tagged polypeptides were expressed in bacteria and purified as
insoluble inclusion bodies. These were used as antigens to induce
antisera in rabbits. Monospecific antibodies were prepared from the
crude antisera by affinity-chromatography (33) using the antigen
immobilized on a SulfoLink gel (Pierce). Affinity-purified antibodies
against individual Mcm proteins have been described (28).
Antibodies were used for immunoprecipitations and for immunoblotting
experiments essentially as originally described (34). The membranes
were developed using the enhanced chemiluminescence system according to
the manufacturer's instructions (ECL, Amersham Pharmacia Biotech).
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RESULTS |
Characterization of Antibodies--
Affinity-purified monospecific
antibodies against hOrc1p and hOrc2p were used to investigate
unfractionated protein extracts prepared at 0.4 M NaCl from
isolated HeLa cell nuclei (Fig.
1A, left). The
hOrc1p-specific antibodies recognized in immunoblots a single
polypeptide band with an apparent molecular weight of ~100 kDa as
expected for hOrc1p (32) whereas the hOrc2p-specific antibodies stained
a polypeptide with an apparent molecular weight of ~67 kDa,
characteristic of hOrc2p (Fig. 1B, right) (32). In several experiments the Orc2 antibodies reacted with two closely spaced hOrc2p-related bands in immunoblots (see below). This has also
been observed with independently prepared hOrc2p-specific antibodies
(28) and could indicate that isoforms of hOrc2p exist in human cells or
that hOrc2p is post-translationally modified.

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Fig. 1.
Characterization of antibodies.
A, immunoblotting. Proteins, extracted at 0.4 M
NaCl from isolated nuclei, were investigated by polyacrylamide gel
electrophoresis and Coomassie staining (left) and blotted
for immunostaining using Orc1-specific and Orc2-specific antibodies as
indicated (right). Asterisk, degradation product
of hOrc2p. Positions of electrophoresis markers are shown at the
left margin. B, immunoprecipitations.
Input, hOrc1p and hOrc2p in unfractionated extracts before
immunoprecipitations. Equal aliquots of the extract were
immunoprecipitated using Orc1-specific antibodies (upper
panel) and Orc2-specific antibodies (lower panel) and
processed for Western blotting.
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As immunoprecipitations are important for the experiments to be
reported below, we demonstrate in Fig. 1B that the
hOrc1p-specific antibodies efficiently precipitated hOrc1p just as the
hOrc2p-specific antibodies precipitated hOrc2p from crude nuclear extracts.
To estimate the amount of hOrc2p per cell we have immunoprecipitated
hOrc2p from a known number of cells and compared the intensities of
Coomassie Blue-stained bands with those of known amounts of recombinant
hOrc2p (not shown). The results suggest about 104 molecules
hOrc2p per cell in asynchronously proliferating HeLa cell cultures.
This indicates that the amount of hOrc2p is at least an order of
magnitude lower than the amount of hMcm3p in proliferating HeLa cell
nuclei (35). hOrc1p could not be detected on the same gel by Coomassie
Blue staining.
hOrc1p and hOrc2p on Chromatin--
As a first attempt to
demonstrate a colocalization of hOrc1p and hOrc2p on chromatin, we
treated isolated HeLa cell nuclei with micrococcal nuclease to separate
different chromatin fractions as originally described by Rose and
Garrard (30) in their study on the nature of chromatin alterations
during lymphocyte development. Briefly, nuclei were incubated with
micrococcal nuclease in the presence of calcium ions, cooled on ice,
and centrifuged to prepare the supernatant fraction (Fig.
2, S1). This fraction has been described to consist of open genetically active chromatin that is
deficient in histone H1 and enriched in high mobility group (HMG) proteins (30). The pellet was resuspended in an EDTA-containing buffer and again centrifuged to prepare the supernatant fraction with
more compact chromatin that contains histone H1 (Ref. 30; Fig. 2,
S2). The remaining insoluble fraction includes the nuclear matrix, but also nuclease-resistant chromatin including actively transcribed gene sequences (Ref. 30; Fig. 2, P). Their
resistance against nucleases may be at least partially because of
associated large protein complexes such as the RNA polymerase
holoenzyme (30) or the human SWI/SNF chromatin remodeling complex which has previously been detected in fraction P chromatin
(36).

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Fig. 2.
Chromatin fractionation. Nuclei from
5 × 106 cells were prepared from exponentially
growing HeLa cells and treated for 10 min at 14 °C in the presence
of 2 mM CaCl2 with 30 units of micrococcal
nuclease (in a 0.1 ml-volume). The supernatant of low speed
centrifugation yielded fraction S1. The pellet was washed
with 8 mM EDTA to yield the supernatant fraction
S2 and the pellet fraction P. A, equal
aliquots from each fraction were deproteinized and analyzed by agarose
gel electrophoresis and ethidium bromide staining. B,
aliquots were investigated by Western blotting using hOrc1p- and
hOrc2p-specific antibodies.
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We have investigated deproteinized aliquots from fractions
S1, S2, and P by agarose gel
electrophoresis. Chromatin fragments in fraction S1 yielded
the familiar ladder of mono-, di-, tri-, and oligonucleosomal DNA with
a strong mononucleosomal band. This band was much less pronounced in
the DNA extracted from fraction S2 as might be expected for
more densely packed chromatin. The DNA extracted from the
nuclease-resistant fraction P migrated as a continuous
spectrum of fragments during gel electrophoresis (Fig.
2A).
Immunoblotting showed that hOrc2p, but not hOrc1p, occurred in the
S1 fraction of highly accessible chromatin (which also contained Mcm proteins; not shown, see Ref. 28; Fig. 2B).
Neither hOrc1p nor hOrc2p could be detected in the more compact
chromatin in fraction S2 (Fig. 2B). In fact, all
hOrc1p and the remaining hOrc2p were found in fraction P
(Fig. 2B), which includes nuclease-resistant chromatin and
the nuclear matrix.
Interestingly, the investigated Orc proteins partition to different
chromatin regions. A considerable fraction of hOrc2p resides on open
chromatin characterized by its accessibility to micrococcal nuclease,
whereas the remaining hOrc2p together with all detectable hOrc1p is
located at nuclear sites that are resistant against nuclease attack.
However, the question of whether hOrc1p and hOrc2p do interact when
bound to chromatin fraction P remains to be answered. Because both hOrc1p and hOrc2p occur in an insoluble fraction under
conditions used in this experiment, immunoprecipitations are impossible
to perform.
We therefore addressed this point by performing cell fractionation
experiments, because we anticipated that stable interactions between
hOrc1p and hOrc2p will be at least partially conserved upon treatment
of chromatin with moderately high salt concentrations.
Briefly, HeLa cells were disrupted in a hypotonic buffer with 5 mM MgCl2 to preserve the integrity of the
nuclear envelope (37). Cytosolic proteins (Fig.
3, Cy) were separated from
nuclei by low speed centrifugation. Subsequently, the nuclei were lysed in 0.5% Nonidet P-40 and centrifuged again to obtain a supernatant with nucleosolic proteins. (Fig. 3, Nu). The residual
nuclear structure was successively eluted with increasing NaCl
concentrations as indicated in Fig. 3.

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Fig. 3.
Differential cell fractionation.
Exponentially growing HeLa cells were used to prepare cytoplasmic
proteins (Cy), nucleosolic proteins (Nu), and an
Nonidet P-40-resistant residual nuclear structure (chromatin and
nuclear matrix), which was successively washed with 100 mM,
250 mM, and 450 mM NaCl. The supernatants were
investigated by Western blotting using the two Orc antibodies and
individual Mcm-specific antibodies (28).
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Both hOrc1p and hOrc2p could only be detected in the nuclei. Most
hOrc2p dissociated at salt concentrations of 0.1-0.25 M NaCl. The 0.25 M NaCl-eluate also contained some hOrc1p.
However, the majority of hOrc1p, together with the remaining
hOrc2p, dissociated at 0.45 M NaCl, a salt concentration
that was also required to mobilize chromatin-bound Mcm proteins (Fig.
3). In this experiment, we used the distribution of Mcm proteins as an
internal control because it is well established that in asynchronously
proliferating HeLa cells, only approximately one-half of the Mcm
proteins is bound to chromatin whereas the other half occurs in the
nucleosol (Refs. 38-40, Fig. 3, Nu and NaCl
eluates). In addition small amounts of Mcm proteins were detected in
the cytosol (Fig. 3, Cy), which most likely originated from
cells in mitosis (41). In fact, no Mcm proteins were detected in the
cytosol of cells arrested at the S/G1 boundary (not shown).
Consistent with the results obtained by chromatin fractionation,
elution of nuclei with NaCl indicates again that there are two
subpopulations of hOrc2p. One that dissociates from chromatin under low
salt conditions and a second fraction that is eluted together with
hOrc1p requiring higher salt concentrations.
Possible interactions between hOrc1p and hOrc2p were investigated by
immunoprecipitation (Fig. 4). From the
0.25 M NaCl extract, Orc1-specific antibodies precipitated
not only the relatively small amounts of hOrc1p present in this extract
but also some associated hOrc2p. Orc2-specific antibodies precipitated
additional amounts of hOrc2p, which were obviously not associated with
hOrc1p (Fig. 4A, left panel). In contrast, both
antibodies precipitated similar amounts of hOrc1p and hOrc2p from the
0.45 M NaCl eluate (Fig. 4A, right
panel) indicating that the interaction between the two proteins
was stable enough to survive the various washing steps required by the
immunoprecipitation protocol. However, in both salt extracts,
Orc2-specific antibodies failed to coimmunoprecipitate a small fraction
of hOrc1p suggesting that this fraction of hOrc1p is not complexed to
hOrc2p.

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Fig. 4.
Coimmunoprecipitation of hOrc1p and
hOrc2p. A, cell fractionation. Nuclear extracts,
prepared at 250 mM and at 450 mM NaCl, were
incubated in parallel with Orc1-specific or with Orc2-specific
antibodies as indicated ( -ORC1 and -ORC2).
Immunoprecipitations were performed and processed for Western blotting.
The blotted proteins were stained with a mixture of Orc1- and
Orc2-specific antibodies. B, chromatin fractionation.
Fraction P chromatin from asynchronous cells (Fig. 2) was
extracted with 0.45 M NaCl for a preparation of
chromatin-bound proteins that were immunoprecipitated with
Orc2-specific antibodies and processed for Western blotting with mixed
Orc1- and Orc2-specific antibodies.
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We wished to demonstrate that the interaction between hOrc1p and hOrc2p
as observed in Fig. 4A could be also detected in the P fraction of nuclease-treated chromatin (see Fig. 2). For
that purpose, we extracted fraction P chromatin with 0.45 M NaCl and analyzed the released protein by
immunoprecipitation. Again Orc2-specific antibodies precipitated not
only hOrc2p but also the hOrc1p present in this fraction (Fig.
4B).
A conclusion from these experiments is that hOrc2p occurs in two
chromatin compartments in human cells. One compartment is characterized
by highly accessible chromatin and contains hOrc2p that dissociates
in vitro at 0.1-0.25 M NaCl. The second
compartment is resistant to nuclease digestion. It contains
hOrc1p/hOrc2p complexes, which are mobilized in buffers with 0.45 M salt.
hOrc1p and hOrc2p in Synchronized Cells--
The experiments
described in the previous section were performed with asynchronously
proliferating HeLa cells. We next performed experiments with cells that
were first arrested by a double-thymidine block at the G1/S
phase transition (29) and then released for a synchronous passage
through S phase and mitosis. DNA synthesis and mitosis were monitored
respectively by incorporation of [3H]thymidine and a
determination of mitotic indices exactly as previously described (28)
(Fig. 5A). Cells were
collected in 1.5-h intervals and processed for chromatin preparation
according to Hancock (31). Aliquots from samples containing equal
amounts of DNA were investigated by Western blotting (Fig.
5B).

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Fig. 5.
Chromatin-bound hOrc1p and hOrc2p in
synchronized HeLa cells. A, cells arrested by a
double-thymidine block were released into S phase by removal of excess
thymidine. Passage through the cell cycle was monitored by the
incorporation of [3H]thymidine, and the microscopic
determination of the fraction of cells in mitosis (mitotic index)
exactly as previously described (28). We present acid-precipitable
[3H]thymidine as a percentage of maximal-incorporated
radioactivity: ~15,000 counts/min at 6 h after release.
B, chromatin according to the procedure of Hancock (31) was
prepared from cells collected at the indicated times. Aliquots
containing equal amounts of DNA were investigated by Western blotting
using a mixture of Orc1- and Orc2-specific antibodies
(upper) and, in a parallel assay, Mcm3-specific antibodies
(lower).
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The results show that large fractions of hOrc2p remained bound to
chromatin during the cell cycle as reported (28). Interestingly, however, the amounts of chromatin-bound hOrc1p decreased during S phase
and early mitosis, but reappeared later in mitosis before the beginning
of a new cell cycle (Fig. 5B). Mcm3p served as a control and
showed the well documented behavior of dissociation during S phase and
reassociation at the end of mitosis (Fig. 5B, Refs.
38-40).
However, although the main portion of hOrc2p remains bound to
chromatin, there is a slight decrease in chromatin-bound hOrc2p correlating with the release of hOrc1p from chromatin during S phase
(Fig. 5B). Taken together with the occurrence of hOrc2p in
two chromatin compartments (see above) the question arises of whether
the population of hOrc2p complexed to hOrc1p was released from
chromatin during S phase.
To address this point we applied the cell fractionation as well as the
chromatin fractionation procedure to synchronously growing S phase
cells (prepared at 6 h after release from a double thymidine block
as in Fig. 5).
First we performed cell fractionation experiments comparing cells
prepared at 4 h after release from a nocodazole block with S phase
cells prepared at 6 h after release from a double thymidine block.
We have determined the cell cycle stages of the cell preparations by
fluorescence-activated cell sorting (FACS) and found in agreement with
others (42), that the cells prepared 4 h after nocodazole release
were entirely in the G1 phase, whereas cells prepared at
6 h after release from a double thymidine block were predominantly in S phase (not shown). As shown in Fig.
6A, 0.1-0.25 M
NaCl mobilized similar amounts of hOrc2p in G1 phase and in
S phase nuclei. However, high salt released hOrc1p and hOrc2p from
G1 phase nuclei but not from S phase nuclei showing that
hOrc1p/hOrc2p-containing complexes are present on G1 phase
chromatin, but not on S phase chromatin. Because hOrc1p could not be
detected in the cytosolic or nucleosolic fractions of Fig.
6A, it must be concluded that hOrc1p was rapidly degraded
after dissociation from chromatin. As a loading control, we eluted
proteins with 2 M instead of 0.45 M NaCl to be
able to detect the core histones in the same fraction as the
hOrc1/hOrc2-complex.

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Fig. 6.
hOrc1p/hOrc2p-complexes dissociate from their
chromatin sites in S phase. HeLa cells synchronized in
G1 phase were prepared 4 h after release from a
nocodazole block (29). S phase cells were prepared 6 h after
release from a double-thymidine block. A, cell
fractionation. Synchronously growing cells in G1 and S
phase were fractionated as described under "Experimental
Procedures" to prepare cytosolic proteins (C), nucleosolic
proteins (N), and chromatin, which was successively treated
with 0.1, 0.25, and 2 M NaCl as indicated. Aliquots with
equal amounts of protein were investigated by SDS-polyacrylamide gel
electrophoresis and Western blotting using Orc1- and Orc2-specific
antibodies (upper panels). The lower part of the same gel
containing the core histones was stained with Coomassie Blue as a
loading control (lower panel). B, chromatin
fractionation. Nuclei were prepared from asynchronously proliferating
cells (asynchron.) and S phase cells. Nuclei were digested
with micrococcal nuclease to prepare chromatin fractions S1
and P (see Fig. 2). Equal aliquots from each preparation
were investigated by Western blotting using a mixture of Orc1- and
Orc2-specific antibodies.
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To confirm the above finding we also performed micrococcal nuclease
digestion experiments comparing nuclei from asynchronously proliferating cells (as in Fig. 2) and from cells in S phase. We
detected similar amounts of hOrc2p in chromatin fraction S1 from asynchronized and S phase cells (Fig. 6B, left
panel). Again hOrc1p/hOrc2p-containing complexes were absent from
chromatin fraction P of S phase cells which contained no
detectable hOrc1p and only small amounts of hOrc2p (Fig. 6B,
right panel).
The conclusions from the experiments shown in Figs. 5 and 6 are that
not only hOrc1p is released from chromatin during S phase but also a
subfraction of hOrc2p, located in the nuclease-resistant chromatin
fraction P, which dissociates together with hOrc1p from chromatin
during S phase. Only hOrc2p associated with nuclease-accessible chromatin remains bound during S phase.
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DISCUSSION |
We have used monospecific antibodies to investigate the
distribution of two components of the human origin recognition complex, hOrc1p and hOrc2p, in HeLa cells. We detected that hOrc1p and hOrc2p
can only be detected on chromatin, but not as free proteins in the
cytosol or in the nucleus. More importantly, most and probably all
chromatin-bound hOrc1p appears to be closely associated with hOrc2p. In
addition, a second fraction of hOrc2p exists that occurs independently
of hOrc1p on chromatin.
These conclusions were supported by two different experiments. First,
differential micrococcal nuclease digestion revealed that a highly
accessible chromatin fraction carries hOrc2p, but not hOrc1p, whereas a
nuclease-resistant chromatin fraction contains both hOrc1p and hOrc2p;
and, second, extraction of isolated chromatin with salt showed that
0.1-0.25 M NaCl sufficed to dissociate free hOrc2p from
chromatin, but >0.45 M NaCl was necessary to release hOrc2p in association with hOrc1p. These results are consistent with
immunoprecipitations of in vivo cross-linked chromatin (28, 43) showing that Orc1-specific antibodies precipitated nucleoprotein fragments carrying both cross-linked hOrc1p and hOrc2p whereas Orc2-specific antibodies precipitate additional nucleoprotein fragments
with cross-linked hOrc2p only (not shown).
Physical interaction between hOrc1p and hOrc2p was demonstrated by
coimmunoprecipitation experiments in agreement with earlier reports
describing that ectopically expressed hOrc1p is able to interact with
hOrc2p in vivo (32). However, others have described that
hOrc1p, extracted from normal proliferating human cells, behaves as a
"single molecule entity" (44) implying that hOrc1p is not
associated with other Orc proteins. We can presently not explain these
differences, but note that the extraction buffer used here contains
ATP, which may stabilize the interaction between Orc proteins (1).
The hOrc1p/hOrc2p-containing complexes, released under our conditions
from chromatin at 0.45 M NaCl, were found to sediment through sucrose gradients at 7-8 S (not shown). This value is clearly
higher than expected for a dimer composed of hOrc1p and hOrc2p only. It
rather indicates that the complex contains additional components (45).
A possibility is that these components may be other human Orc proteins
in analogy to the free heterohexameric ORCs detected in extracts from
Xenopus eggs and Drosophila embryos (19, 46).
This possibility will be investigated once reliable antibodies against
other human Orc proteins become available.
HOrc1p/hOrc2p-containing complexes are located in chromatin regions
that are resistant against nuclease digestion. Nuclease-resistant chromatin is frequently assumed to be a component of the nuclear matrix. However, the nuclear matrix is operationally defined as a
structure that remains after extensive nuclease digestion and treatment
with high salt (47). Accordingly hOrc1p/hOrc2p-containing complexes may
be associated with the nuclear matrix, but they cannot be integral
components of the matrix, because they are released from the
nuclease-resistant fraction by moderately high salt concentrations.
They share this property with transcribing RNA polymerase (30) and the
human SWI/SNF chromatin remodeling complex (36), two large protein
assemblies, which have also been found in nuclease-resistant chromatin.
It has to be investigated whether large protein complexes may aggregate
stretches of chromatin and thereby protect them against nuclease attack.
hOrc2p is not only a partner of hOrc1p, but occurs also independently
of hOrc1p on chromatin. An implication is that the amounts of hOrc2p in
HeLa nuclei must be higher than the amounts of hOrc1p. However, it is
presently not possible to express this difference in reliable numbers
because Western blot analyses are not quantitative and are difficult to
compare as neighboring or overlapping protein bands may affect the
intensities of the immunostains. A rough estimate, based on
immunostainable bands in salt-released chromatin proteins, suggests a
2-3-fold higher amount of hOrc2p than hOrc1p in HeLa cell chromatin.
But these figures may need to be modified once the proteins are
isolated as pure entities.
What is the role of hOrc2p that occurs independently of hOrc1p in the
nuclease-sensitive chromatin fraction? It is known that Orc proteins
have tasks "beyond replication" (see review in Ref. 48). In
particular, Orc2p has been shown to be involved in the organization of
heterochromatin in Drosophila (49-52), just like yeast Orc
proteins are known to have functions in transcriptional silencing and
mitosis (49-53). If human hOrc2p performed similar functions, it would
be expected to occur in more compact chromatin regions. This is clearly
not the case, and further investigations on hOrc2p in extended
chromatin are necessary.
In either case, the fraction of hOrc2p in extended chromatin appears to
have functions distinct from the fraction of hOr2p in complex with
hOrc1p. We conclude this because uncomplexed hOrc2p remains stably
bound to chromatin in contrast to hOrc1p/hOrc2p-containing complexes,
which leave their chromatin sites when HeLa cells pass through S phase
and reappear soon after mitosis with an entry into a new cycle. This
was shown here by experiments with cells synchronized by a double
thymidine block (Figs. 5 and 6), but we obtained consistent data
studying HeLa cells after release from a nocodazole-induced mitotic
block. Under these conditions, hOrc1p could be detected on chromatin at
3-5 h after removal of nocodazole (Fig. 6A, left
panel). Thus, the important conclusion is that
hOrc1p/hOrc2p-containing complexes must undergo drastic structural
changes during S phase, which eventually lead to their dissociation
from chromatin and the loss of hOrc1p during S phase.
Our data is in line with results obtained with other systems. As
mentioned in the Introduction, xORC dramatically changes its structure
after formation of the prereplication complex (22-24, 54), and
experiments on the replication capacity of hamster cell nuclei in
Xenopus egg extracts gave clear evidence for a de
novo assembly of functional hamster cell ORCs early in
G1 phase (27).
Presently, the fate of released hOrc1p in cycling HeLa cells remains
unclear. We have no evidence for released hOrc1p in the cytosolic or
nucleosolic fraction suggesting that released hOrc1p may be rapidly
degraded. In this case, hOrc1p must be synthesized de novo
at the beginning of each cell cycle. This would be consistent with the
finding that the human gene encoding hOrc1p is activated in a cell
cycle-dependent manner in contrast to the hOrc2p-encoding gene, which is constitutively active (55). In fact, the fate of hOrc2p
in the hOrc1p/hOrc2p-containing complex is uncertain. A possibility is
that it enters the pool of the hOrc1p-independent hOrc2p fraction in
extended chromatin. This is in clear contrast to the behavior of Mcm
proteins, which also dissociate from chromatin in a
replicationdependent manner, but accumulate as free proteins in
the nucleosol and rebind to chromatin at the beginning of the following
cell cycle.
In summary, we have shown here that the hOrc1p/hOrc2p complex,
components of the human replication initiation machine, do not remain
on their chromatin binding sites during S phase and mitosis. This could
be a powerful mechanism to prevent the rereplication of already
replicated genome sections within a cell cycle. Our preliminary
evidence suggests that hOrc1p is degraded during S phase and
synthesized de novo when cells enter a new cell cycle. An
important consideration for further research are the mechanisms by
which these events are coordinated within a cell cycle.