The Human Origin Recognition Complex Protein 1 Dissociates from Chromatin during S Phase in HeLa Cells*

Sandra KreitzDagger §, Marion RitziDagger , Martina BaackDagger , and Rolf KnippersDagger

From the Dagger  Department of Biology, Universität Konstanz, D-78457 Konstanz and the  GSF-Haematologikum, Marchioninistrasse 25 D-81377 München, Germany

Received for publication, October 17, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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).

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 (alpha -ORC1 and alpha -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.

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).

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank Claudia Gruss and Frank O. Fackelmayer for discussions and critical reading.


    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 49-7531-88 2127; Fax: 49-7531-88 4036; E-mail: Sandra.Kreitz@uni-konstanz.de.

Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M009473200


    ABBREVIATIONS

The abbreviations used are: ORC, origin replication complex; Mcm, minichromosome maintenance.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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