SUMMARY
The composition of the origin recognition complex (ORC with its components hOrc1p-hOrc6p) was investigated in synchronously proliferating human HeLa cells.
Chromatin prepared from cells in the G1-phase of the cell cycle was found to carry two major forms of ORC, one with proteins hOrc1p-hOrc5p, and a second one lacking hOrc1p. Neither form of ORC contained hOrc6p of which most was found in the soluble fraction of nuclear proteins. Chromatin from cells in late S-phase only contained the hOrc2p-hOrc5p form of ORC, because hOrc1p was gradually released from chromatin during S-phase followed by its rapid destruction. Metabolic labeling experiments supported this concept and showed that the entire amount of hOrc1p was newly synthesized during the G1-phase of each cell cycle. The S-phase-dependent release of hOrc1p from chromatin was accompanied by its phosphorylation, which appears to be triggered by the establishment of active replication forks. This was concluded because aphidicolin, a potent inhibitor of replicative DNA-polymerases, prevented the release of hOrc1p from chromatin. Interestingly, inhibitors of proteasome-dependent proteolysis also blocked DNA replication and consequently the release of hOrc1p from chromatin.
INTRODUCTION
The ordered replication of eukaryotic genomes requires the formation of functional pre-replication complexes that assemble in a stepwise manner during the G1-phase of the cell cycle. The assembly begins with the six-subunit origin recognition complexes (ORC with its constituents Orc1p-Orc6p) and continues with the recruitment of a number of additional replication factors including Cdc6p, Cdt1p and the six MCM proteins, Mcm2p-Mcm7p. Once formed, pre-replication complexes become activated at the beginning of S-phase by at least two types of kinases, cyclin-dependent kinases and the Dbf4-dependent Cdc7-kinase. This activation is required for the association of additional proteins, which eventually lead to the establishment of replication forks (recently reviewed in Bell and Dutta (2002), Ellison and Stillman (2001) and Nishitani and Lygerou (2002)).
ORC was originally discovered as an essential protein complex that binds in an ATP-dependent manner to a conserved sequence element in yeast replication origins (Bell and Stillman, 1992). Subsequent studies have shown that the six ORC proteins are conserved among all eukaryotes, where they form the basis for pre-replication complex assembly as outlined above (Bell and Dutta, 2002; Chesnokov et al., 1999; Coleman et al., 1996; Dhar et al., 2001a; Landis et al., 1997; Pinto et al., 1999; Romanowski et al., 1996; Rowles et al., 1996). However, while the structure and function of ORC seem to be generally well conserved among eukaryotes, more detailed studies reveal interesting differences between yeast and metazoan systems. For example, yeast ORC remains stably bound to chromatin during the cell cycle (Aparicio et al., 1997; Liang and Stillman, 1997), but Xenopus ORC proteins loosen their contacts with chromatin sites after the loading of MCM proteins, and can be removed without affecting DNA replication (Hua and Newport, 1998; Rowles et al., 1996). Similarly, the largest subunit, Orc1p, could be found in ORC of actively proliferating Drosophila cells, but not in ORC of resting Drosophila cells (Asano and Wharton, 1999; Royzman et al., 1999).
Cell cycle-dependent variations in ORC have also been detected in mammalian cells in culture. A first observation was that nuclei prepared from hamster cells in early G1-phase required exogenous Orc1p for replication in Xenopus egg extract, whereas nuclei from late G1-phase cells did not, implying that early-G1-phase ORCs lack Orc1p, and more generally, that functional ORCs assemble as cells progress through G1-phase (Natale et al., 2000). Indeed, work with synchronized human HeLa cells indicated that hOrc1p is gradually released from chromatin during S-phase, degraded and then resynthesized in the following cell cycle (Kreitz et al., 2001). The cell cycle-dependent degradation of mammalian Orc1p is controlled by the SCF ubiquitin-ligase complex (Mendez et al., 2002) and requires ubiquitination (Li and DePamphilis, 2002;
Mendez et al., 2002; Sun et al., 2002) for proteasome-mediated destruction. The regulated destruction of mammalian Orc1p is considered to be a powerful mechanism to prevent the re-replication of genome sections that had already been replicated during the same S-phase (Cimbora and Groudine, 2001).
In our previous paper we demonstrated that two forms of ORC might occur in HeLa cells. One form contains hOrc1p and is bound to chromatin in a manner that requires higher salt concentrations for dissociation than the second form that lacks hOrc1p. This conclusion depended on the results with two ORC proteins, hOrc1p and hOrc2p. As the S-phase-dependent dissociation of Orc1p changes the composition of mammalian ORC, it appeared necessary to also determine the behavior of the remaining ORC proteins in synchronized human cells. We demonstrate here that the dissociation of hOrc1p during S-phase is an important reaction affecting the structure of ORC, and have investigated conditions for its dissociation and cell cycle-dependent destruction as well as the rate of hOrc1p synthesis in the subsequent cell cycle. Interestingly, the dissociation of hOrc1p is apparently coupled to DNA replication, and possibly depends on the progression of replication forks.
EXPERIMENTAL PROCEDURES
Cell Culture, Synchronization and FACS-Analysis
HeLa S3 and Cos7 cells were grown on plastic dishes in Dulbecco’s modified Eagle’s medium (DMEM) plus 5% fetal calf serum in a humidified atmosphere containing 5%
CO2.
HeLa cells were synchronized either by a double-thymidine block at the beginning of S-phase and released into thymidine-free medium as described before (Krek and DeCaprio, 1995; Ritzi et al., 1998) or by nocodazole treatment at mitosis. For this purpose, exponentially growing cells were pre-synchronized by a single thymidine block (2 mM thymidine for 14 h) and then released into thymidine-free medium. After about 7 h 40 ng/ml nocodazole was added to the cells for another 4 h before the medium was removed and mitotic cells were shaken off the plates.
For FACS analysis, plates were washed three times with phosphate-buffered saline (PBS; 130 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) plus 5 mM EDTA and kept for 30 min at 4°C before cells were resuspended in PBS/EDTA.
4x105 cells were pelleted (600 g, 5 min) and resuspended in 300 µl staining solution (PBS + 5 mM EDTA, + 1% TritonX-100, + 15 µg propidium-iodide). The samples were analyzed after 30 min on ice (FACS-Calibur and Cell Quest, BD Bioscience).
The progression through S-phase was monitored by measuring the rate of DNA synthesis. To this end, 3x106 cells were released from a double-thymidine block into S-phase, and were labeled for 1 h in consecutive 1 h-intervals with 1 µCi/ml [3H]-thymidine (2 Ci/mmol, Amersham) before cells were harvested. After washing twice with PBS, cells were lyzed in 2.5 ml PBS containing 2% SDS for 1 h followed by dilution with PBS (1:20–to reduce DNA viscosity) and TCA precipitation (Maniatis et al., 1987) of 2 ml-aliquots of the lysates.
Cell Fractionation, Total Cell Extract and S20-Extract
Cell fractionation was performed as described (Kreitz et al., 2001). 2-3x107 cells were washed three times with PBS, scraped off the plates and suspended in 5 ml 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 (1000 g, 5 min, 4°C) to separate the cytosolic supernatant from the nuclear pellet. Nuclei were resuspended in 5 ml buffer A with 0.5% Nonidet-P40 (NP40) and kept on ice for 15 min to lyze the nuclear envelope. Centrifugation (1000 g, 5 min, 4°C) separated the free nucleosolic proteins from the pelleted nuclei. The pellet was eluted (15 min on ice) with 5 ml 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. To prevent protein degradation, both buffers A and B were
supplemented with protease inhibitor (Complete EDTA-free, Roche Molecular Diagnostics) and the proteasome inhibitor MG-132 (10 µM).
To prepare total cell extract, 2-3x107 cells were washed three times with PBS, scraped off the plates and collected by centrifugation (600 g, 5 min, 4°C). The pellet was resuspended in 5 ml 2% SDS, followed by sonication for 3x10 sec (70 W). Proteins were concentrated using the method of Wessel and Flugge (1984) before they were analyzed by denaturing polyacrylamide gel electrophoresis.
To obtain S20-extract, 6x108 HeLa cells were washed twice with PBS, scraped off the plates and collected by centrifugation (164 g, 5 min, 4°C). The pellet was suspended in 10 ml hypotonic buffer (10 mM HEPES, pH 7.5; 5 mM KCl; 1.5 mM MgCl2; 0.1 mM DTT) plus 250 mM sucrose. After a washing step with 10 ml hypotonic buffer without sucrose, cells were kept on ice for 10 min followed by dounce homogenization (12x).
After a further 30 min on ice, the lyzed cells were centrifuged for 1 h at 20,000 g (4°C).
The supernatant, containing the soluble proteins from cytoplasm and nucleoplasm, was divided into aliquots and frozen at -70°C. Protein concentration was determined using the Bio-Rad Protein Assay Kit (Biorad) according to the manufacturer’s instructions.
The average protein concentration was 6-12 µg|µl.
Expression Plasmids
A plasmid expressing T7-epitope-tagged hOrc1p under the control of a CMV-promotor (pKG28) was kindly provided by B. Stillmann (Cold Spring Harbor Laboratory).
pRSET-hOrc1p was created by PCR-amplification of the ORF of hOrc1p and subcloning into pRSET B (Invitrogen) using the restriction sites BamHI and HindIII.
The plasmid expressing His6- and myc-tagged ubiquitin (pCW7) was a gift from J. Falck and J. Bartek (Danish Cancer Society, Copenhagen, Denmark).
pVL1393-GST-Cdc6 [E285Q], kindly provided by E. Fanning (Vanderbilt University, Nashville, Tennessee, USA), encodes for full-length GST-hCdc6p.
Proteasome Inhibition and In Vivo Ubiquitination Assay
For inhibition of the proteasome activity, exponentially growing cells were treated for 13 h with Lactacystin, ALLN or MG-132 (all from Calbiochem) at the indicated concentrations. Equal amounts of cells were used to prepare structure-bound proteins eluted at 450 mM NaCl (see Cell Fractionation).
Detection of ubiquitinated intermediates in Cos7 cells was achieved according to the method of Treier and coworkers (Treier et al., 1994) with the modification that the TCA precipitation was replaced by the precipitation according to Wessel and Flugge (1984).
1x106 cells were transfected with the indicated amounts of plasmid DNA for the overexpression of T7-hOrc1p (pKG28) and His6- and myc-tagged ubiquitin (pCW7) and 46 h posttransfection cells were used for isolation of in vivo-ubiquitinated proteins.
Transfection
Transfection was performed according to the method of Boussif et al. (1995) with minor modifications. A 0.45% working solution was freshly prepared from a 25% stock solution of water-free polyethylenimine (PEI) (Sigma/Aldrich). For each 94 mm plastic dish 4.5 µl of the working solution was added to 300 µl of serum-free medium and in a separate tube 10 µg of DNA was diluted into 50 µl serum-free medium. The diluted PEI was added to the DNA and the mixture was immediately vortexed for 5 sec and incubated for 30 min at RT. Culture medium was removed and 3 ml fresh medium was added before the PEI/DNA mixture was added dropwise to the cells. After mixing, the plates were incubated for 4 h before the medium was replaced by normal culture medium. When more than 10 µg of DNA was used for transfection, the amount of PEI was adjusted to maintain a ratio of 15:1 (moles nitrogen versus moles phosphorus in PEI and DNA, respectively).
In Vivo Labeling with [35S]-Methionine
To monitor the synthesis of hOrc1p across the cell cycle, HeLa cells were seeded with a density of 2x106 cells per 94 mm plastic dish and synchronized by a double-thymidine block. Excess thymidine was removed for release into the cell cycle and plates were labeled during consecutive 3 h-intervals. After washing 3 times with methionine-free medium (Gibco), cells were incubated with 50 µCi/ml [35S]-methionine (1175 Ci/mmol, ICN) in methionine-free medium for 3 h before cells were used for cell fractionation and immunoprecipitation.
For pulse-chase experiments, cells were synchronized using nocodazole. Mitotic cells were seeded with a density of 8x106 cells per 94 mm plastic dish in methionine-free medium containing 50 µCi/ml [35S]-methionine (1175 Ci/mmol, ICN) and incubated for 4 h before cells were washed twice with regular medium and then cultured in fresh medium containing 10x methionine.
In Vivo Labeling with [32P]-Ortho-Phosphate
HeLa cells were seeded and synchronized as described above. For labeling, plates were washed twice with pre-warmed SSC-buffer (0.15 M NaCl, 15 mM sodium citrate, pH 7.4) and once with phosphate-free medium (ICN) prior to cultivation in 4 ml phosphate-free medium but supplemented with 50 µCi/ml [32P]-ortho-phosphate (carrier free, ICN) and 5 µM MG-132 (Calbiochem).
In Vitro Transcription-Translation and In Vitro Phosphorylation Assay
The in vitro transcription-translation reaction was performed using the T7-TNT-Coupled Reticulocyte Lysate System (Promega) and the expression plasmid pRSET-hOrc1p according to the manufacturer’s conditions.
Recombinant GST-hCdc6p was expressed in Hi5-cells using the Baculovirus expression vector pVL1393-GST-Cdc6 [E285Q] according to Herbig et al. (1999).
For in vitro phosphorylation assays, 5 µl of in vitro-translated hOrc1p or 300 ng of recombinant GST-hCdc6p were incubated with 145 µg S20-extract, 1 mM sodium vanadate, 25 mM NaF, protease inhibitor (Complete EDTA-free, Roche Molecular Diagnostics) and 30 µCi γ[32P]-ATP (>4,000 Ci/mmol, ICN) at 25°C. Reactions were stopped after indicated times by the addition of 10 mM EDTA, 450 mM NaCl, 0.5% NP40 and 20 mM ATP and storage on ice. hOrc1p was recovered by immunoprecipitation, while GST-hCdc6 was purified using glutathione agarose (Sigma). Samples were subjected to SDS-PAGE, and western blotted. For detection of the labeled protein, the dried blotting membrane was used for exposure, followed by the detection of the input protein by western analysis with specific antibodies against Orc1p or Cdc6p.
For inhibition of cyclin-dependent kinases S20-extract was preincubated with 500 nM Cip1 (kindly provided by U. Strausfeld, University of Konstanz) for 10 min on ice before the reaction was started (15 min at 25°C, see above).
Antibodies and Immunoblotting
Antibodies against human Orc1p and Orc2p (Kreitz et al., 2001) and antibodies against human Orc3p-Orc6p (Keller et al., 2002) have been described before. Monospecific antibodies were prepared from the crude antisera by affinity-chromatography (Harlow and Lane, 1988) using the antigen immobilized on a SulfoLink gel (Pierce). Affinity-purified antibodies against individual hMCM proteins have been described (Ritzi et al., 1998). Antibodies against cyclin A and cyclin E were purchased from Oncogene.
Antibodies were used for immunoprecipitations and for immunoblotting experiments essentially as originally described (Towbin et al., 1979). The membranes were developed using the enhanced chemiluminescence system according to the manufacturer’s instructions (ECL; Amersham Pharmacia Biotech).
RESULTS
ORC Proteins on Chromatin
In a previous communication, we have described experiments on the chromatin-association of hOrc2p in proliferating human HeLa cells. We found that in G1-phase, hOrc2p is bound to chromatin in two different modes. The first is characterized by its high accessibility to micrococcal nuclease and sensitivity to relatively low salt concentrations, whereas the second is nuclease-resistant and requires higher salt for dissociation. Interestingly, only hOrc2p in this second, salt-resistant mode of chromatin binding was found to be associated with hOrc1p. Consequently, when hOrc1p dissociated from chromatin in S-phase, essentially all hOrc2p appeared in the nuclease-and salt-sensitive first binding mode (Kreitz et al., 2001).
We were interested to know how the other human ORC proteins, hOrc3p-hOrc6p, behaved under these conditions. In a first experiment, we fractionated asynchronously proliferating HeLa cells and separated the soluble (cytosolic and nucleosolic) fractions from a nuclear pellet. The nuclear pellet was treated with buffers of increasing ionic strength to mobilize chromatin-bound proteins. The various protein fractions were analyzed by polyacrylamide gel electrophoresis and immunoblotting using antibodies against all six human ORC proteins.
We determined that hOrc1p-hOrc5p exclusively occurred in the chromatin fraction, in contrast to hOrc6p of which high amounts appeared in the soluble nucleosolic and cytosolic fractions. The presence of hOrc6p in the cytosolic fraction could be due to leakage from nuclei during preparation, but could also arise from cells in mitosis when hOrc6p appears to be involved in chromosome partitioning and cytokinesis (Prasanth et al., 2002).
The chromatin-bound ORC proteins were eluted in buffers with 0.1-0.45 M NaCl. Salt concentrations of 0.1-0.25 M were sufficient to elute substantial amounts of hOrc2p-hOrc6p. However, most hOrc1p together with the remaining amount of hOrc2p-hOrc5p required higher concentrations of NaCl (0.32-0.45 M) for dissociation from the chromatin pellet (FIG 5-1A).
Cell fractionation experiments were then performed with synchronously proliferating HeLa cells in the G1-phase and the S-phase of the cell cycle. The results with G1-phase cells showed again that one fraction of hOrc2p-hOrc6p could be mobilized from chromatin at relatively low salt, while a second fraction of hOrc2p-hOrc6p together with hOrc1p required higher salt for mobilization (FIG 5-1B, left). In S-phase cells, almost all hOrc1p had disappeared, and all ORC proteins appeared in the low salt fraction (FIG 5-1B, right).
FIG 5-1 Distribution of ORC proteins in cell extracts. (A) Exponentially growing cells were used for the preparation of cytoplasmic proteins (C), nucleosolic proteins (N) and a remaining NP40-resistant structure, which was extracted with increasing salt concentrations as indicated. Identical amounts of proteins of each fraction were investigated by western blotting using specific antibodies against all six subunits of the human ORC-complex. (B) HeLa cells synchronized in G1-phase were prepared at 4 h after release from nocodazole block. S-phase cells were prepared at 6 h after release from a double-thymidine block. Cell fractionation and western blotting was performed as above.
Thus, the results of FIG 5-1 extend previous findings (Kreitz et al., 2001) and suggest that ORC occurs in two different forms on G1-phase chromatin: one form contains hOrc1p and requires 0.45 M NaCl for dissociation from chromatin, while the second form lacks hOrc1p and dissociates from chromatin at low salt. In S-phase cells, the hOrc1p-containing form of ORC is absent and the remaining ORC proteins elute from chromatin at 0.1-0.25 M NaCl.
For further investigations, we centrifuged the high- and low-salt forms of ORC through sucrose gradients. We determined that most hOrc1p in the high salt extracts sedimented in a complex of 9-11 S together with hOrc2p-hOrc5p. Small amounts of slower sedimenting forms were also detected, characterized by prominent hOrc4p/hOrc5p bands and a single hOrc1p band (FIG 5-2A, upper). Centrifugation of the low-salt
chromatin extracts resulted in a separation of a faster moving hOrc2-5p complex from slower moving ORC protein subcomplexes (FIG 5-5-A, lower).
Co-immunoprecipitation experiments confirmed the centrifugation results. We incubated high salt extracts with antibodies against hOrc1p, hOrc2p or hOrc4p and found that each one of these antibodies precipitated complexes containing hOrc1p-hOrc5p (FIG 5-2B, upper left). Note that hOrc5p is difficult to detect because it migrates close to the IgG heavy chain in polyacrylamide gels. However, inspection of the supernatants showed that hOrc1p-antibodies could precipitate essentially all of hOrc1p, but not all of hOrc2p-hOrc5p, showing that the extract contained a form of ORC that lacked hOrc1p (FIG 5-2B, lower left). This result and the presence of free hOrc1p in sucrose gradients could indicate that a fraction of hOrc1p dissociated from ORC during preparation. This is in agreement with published reports showing that hOrc1p may be rather loosely bound to a hOrc2-5p core complex (Dhar et al., 2001a;
Vashee et al., 2001).
FIG 5-2 Interactions between ORC proteins. (A) Sucrose gradient centrifugation. Differential cell fractionation was performed with asynchronously growing HeLa cells as described in experimental procedures. Supernatants of the 250 mM NaCl and the 320 mM NaCl elution step were applied to a sucrose gradient (4.5 ml, 10-50% sucrose in 20 mM HEPES, 250 mM NaCl, 0.5 mM MgCl2, 1 mM ATP, 0.3 M sucrose; pH 7.5). Centrifugation was performed in a Beckman SW55 rotor at 4°C for 16 h at 50,000 rpm. Fractions were analyzed by immunoblotting with antibodies against the individual hORC subunits. Arrow, direction of centrifugation. (B) Immunoprecipitations. HeLa cells synchronized in late G1-phase were prepared at 18 h after release from a double-thymidine block. Proteins extracted at 250 mM and 320 mM NaCl from isolated nuclei were used for immunoprecipitations with Orc1p-, Orc2p- or Orc4p-specific antibodies. Eluates (upper panel) and supernatants (lower panel) were processed for western blotting. About 2.5 times more of the 320 mM extract was used compared to the 250 mM extract.
Immunoprecipitations of the low salt chromatin extract are shown in the right panels of FIG 5-2B. Antibodies against hOrc1p failed to precipitate ORC proteins, as expected since low salt chromatin extracts did not contain hOrc1p. However, hOrc2p- and hOrc4p-specific antibodies precipitated the remaining ORC proteins (upper panel). The supernatants of the immunoprecipitations were also informative, showing that Orc2p-antibodies efficiently depleted the extract of hOrc2p and hOrc3p, while significant amounts of hOrc4p and possibly also of hOrc5p remained in the supernatant. Likewise, Orc4p-antibodies efficiently removed hOrc4p and to a lesser extent the other ORC proteins, of which considerable fractions remained in the supernatant (lower panels).
Immunoprecipitations of the low salt chromatin extract are shown in the right panels of FIG 5-2B. Antibodies against hOrc1p failed to precipitate ORC proteins, as expected since low salt chromatin extracts did not contain hOrc1p. However, hOrc2p- and hOrc4p-specific antibodies precipitated the remaining ORC proteins (upper panel). The supernatants of the immunoprecipitations were also informative, showing that Orc2p-antibodies efficiently depleted the extract of hOrc2p and hOrc3p, while significant amounts of hOrc4p and possibly also of hOrc5p remained in the supernatant. Likewise, Orc4p-antibodies efficiently removed hOrc4p and to a lesser extent the other ORC proteins, of which considerable fractions remained in the supernatant (lower panels).