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Originally published In Press as doi:10.1074/jbc.M600299200 on February 22, 2006

J. Biol. Chem., Vol. 281, Issue 16, 10926-10934, April 21, 2006
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Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts*

Shou Waga{ddagger}1 and Akiko Zembutsu§

From the {ddagger}Laboratories for Biomolecular Network, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871 and the §Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan

Received for publication, January 11, 2006 , and in revised form, February 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23 °C, with one or a few origin recognition complex (ORC) molecules and ~10–20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with ~10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions ~10–20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0 °C) without geminin, with ~10–20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23 °C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas ~10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2–7 complexes during metazoan pre-RC formation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA replication in eukaryotes is initiated through the coordinated actions of replication initiation proteins. Previous studies have identified the proteins that are involved in the initiation step and an outline of the mechanism of DNA replication initiation in eukaryotic cells has been described (1–3). In brief, the origin recognition complex (ORC),2 which consists of six subunits (ORC1–6), binds to DNA and then, in cooperation with CDC6 and CDT1, loads the mini-chromosome maintenance (MCM) 2–7 protein complex onto DNA to form a specific protein-DNA complex; the so-called pre-replicative complex (pre-RC). The pre-RC is formed in a cell cycle-regulated manner that involves geminin, a cell cycle-regulated protein that acts as an inhibitor of MCM loading (2, 4). Subsequent activation of the pre-RC is required for unwinding of the DNA around the origin and the start of DNA synthesis (1, 5–11).

ORC and most other initiation proteins are well conserved in various eukaryotic species (1). Saccharomyces cerevisiae ORC binds specifically to the autonomously replicating sequence, a DNA sequence functioning as a replicator in DNA replication (12). Fission yeast Schizosaccharomyces pombe ORC contains AT-hook motifs within the ORC4 subunit and thus binds preferentially to A/T-rich sequences that are common in S. pombe origins (13). In contrast, ORC in metazoans such as humans, Drosophila, and Xenopus does not exhibit sequence specificity in its DNA binding (14–18), but DNA topology has been shown to affect Drosophila ORC-DNA affinity (19). These differences in sequence specificity of ORC-DNA binding partly reflect the different organization of replication origins in the genomes of these species.

ORC1, -4, and -5, CDC6, and MCM2–7 are all members of the AAA+ ATPase family (20, 21). In addition, it has recently been proposed that ORC2 and -3 also belong to this family (22). Previous studies have shown that ATP binding and hydrolysis by the ORC subunits and CDC6 are necessary for pre-RC formation (23–29). S. cerevisiae ORC and CDC6 cooperatively bind to autonomously replicating sequence in an ATP-dependent manner, and it has been suggested that the ORC interaction with both CDC6 and autonomously replicating sequence activates ORC, leading to pre-RC formation (22). However, the precise mechanism of pre-RC formation remains obscure.

During and after pre-RC activation, the MCM2–7 complex is thought to function as a DNA helicase at the origin and also at the replication fork during elongation (30–36). Based on the crystal structure of an archaeal MCM homohexamer and electron microscopic analyses of the S. pombe MCM2–7 complex and the human MCM4-6-7 sub-complex, MCM2–7 and the MCM4-6-7 sub-complex are thought to form a ring-shape structure (37–39). Although mammalian MCM4-6-7 sub-complexes exhibit helicase activity (31, 40), the nature of the active form of the MCM complex during DNA replication remains unclear.

Chromatin-immunoprecipitation analyses of S. cerevisiae MCM have revealed specific association of MCM with both the origin and replication forks (32). In contrast, MCM proteins appear to be widespread over chromatin before the onset of DNA replication in mammalian cells or Xenopus reconstituted nuclei (41–43). Furthermore, MCM proteins are not colocalized with sites of DNA replication in nuclei of mammalian cells or in reconstituted nuclei in Xenopus egg extracts (42–44). Moreover, it has been shown that the MCM2–7 complex loaded onto chromatin is 20–40 times more abundant than ORC bound to chromatin before the onset of DNA replication in Xenopus egg extracts (45–47). These contradictory observations for metazoan MCM proteins, referred to as the "MCM paradox," suggest that the MCM2–7 helicase is more than a simple DNA helicase (44).

Analysis of the in vitro assembly of S. cerevisiae pre-RC has suggested that ORC may load at least two MCM2–7 complexes at the origin (24). During pre-RC formation in Xenopus egg extracts, it has also been suggested that multiple copies of the MCM2–7 complex can be repeatedly loaded onto DNA once ORC is bound to the DNA (46). However, it remains unclear how, in the presence of assembled nucleosomes, such multiple MCM2–7 complexes can be loaded and slide along DNA such that they are widely distributed over the chromatin.

To obtain insights into the mechanism of metazoan pre-RC formation, in this study we analyzed de novo pre-RC formation on naked circular plasmids in Xenopus egg extracts, using an assay that we have recently developed.3 We found that the ORC-CDC6-CDT1-DNA complex is significantly stabilized if the subsequent MCM loading step is inhibited by either the addition of geminin or incubation at decreased temperature. Under these MCM-inhibitory conditions, multiple copies of ORC and CDC6 were bound to each plasmid, but upon MCM loading the ORC-CDC6-CDT1-DNA complex dissociated, leaving multiple copies of MCM complexes and only one or a few ORC molecules on each plasmid. The estimated numbers of these proteins suggest that only one or a few molecules of MCM2–7 complexes are loaded per ORC-DNA binding event, and this provides the basis for discussion of a possible mechanism for loading of multiple MCM complexes in metazoans.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—pG5{lambda}6.6 (11 kb) is a plasmid based on the pGEM-7Zf(+) plasmid (Promega). It contains five Gal4 binding sites, an adenovirus major late promoter, and a 6.6-kb {lambda} DNA/HindIII fragment. pKS-EX (5 kb) is a pBluescript KS-based plasmid that contains a 2.2-kb Epstein-Barr virus ori P sequence (a gift from Dr. Masaki Shirakata, Tokyo Medical and Dental University, Japan). pBlue{lambda}2.0 (5 kb) was constructed by inserting a 2.0-kb HindIII fragment of phage {lambda} DNA into the HindIII site in pBluescript SK(–) (Stratagene). pKS{lambda}7.2 (10 kb) was constructed by inserting a 7.2-kb BamHI fragment of {lambda} DNA into the BamHI site in pBluescript KS(–). pEX{lambda}6.6 (12 kb) was constructed by inserting a 6.6-kb HindIII fragment of {lambda} DNA into the HindIII site in pKS-EX. These plasmids were purified using a Genopure Plasmid kit (Roche Applied Science) and used for preparation of the plasmid beads, as described below.

Biotinylation of Plasmids and Binding to Superparamagnetic Beads—Circular plasmid DNA was biotinylated by photocoupling with Photoprobe (S-S) Biotin (Vector Laboratories), which has a cleavable disulfide bond in the linker arm. The photocoupling reaction was performed according to the manufacturer's instructions. Briefly, 100 µg (200 µl in sterile water) of plasmid was mixed with 0.5 µg (0.5 µl) of photoreactive biotin, and the mixture was irradiated on ice for 10 min at a distance of 5 cm with a 365 nm UV lamp (8 watts). The mixture was then mixed with an equal volume of 0.1 M Tris-HCl (pH 9.5) and subjected to 2-butanol extraction to remove free photoreactive biotin. The biotinylated plasmids were precipitated with ethanol, dissolved in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA (~0.25 mg/ml DNA), and stored at –20 °C. These plasmids (10 µg) were used for binding to 400 µg of streptavidin-Dynabeads M-280 (Dynal Biotech), with the binding reaction carried out with a Dynabeads kilobaseBINDER kit (Dynal Biotech) according to the manufacturer's instructions. The binding reaction (in a siliconized 1.5-ml tube) was performed overnight at 24 °C with constant gentle agitation. After binding, the beads were washed three times with 2 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA, resuspended in the same washing buffer (10 mg/ml beads), and stored at 4 °C. Typically, ~60 ng of circular plasmid was coupled to 10 µg of beads.

Egg Extracts and Recombinant Protein—Xenopus eggs were obtained from a chorionic gonadotropin-injected female frog. The eggs were dejellied in 2% (w/v) cysteine and then rinsed three times in MMR (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA, and 5 mM HEPES, pH 7.8) (48) at room temperature. Dejellied eggs were activated by incubating in 0.5 µg/ml calcium ionophore A23187 [GenBank] (in MMR) for ~5 min at room temperature, and then rinsed five times in ice-cold XB (100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 10 mM HEPES-KOH (pH 7.7), and 50 mM sucrose) (48). Activated eggs were transferred in SW55 tubes (Beckman) that had previously contained 1 ml each of ice-cold XB supplemented with 100 µg/ml cytochalasin B and 10 µg/ml each of aprotinin, leupeptin, and chymostatin, and then packed by centrifuging at 170 x g for 1 min. After excess buffer was removed, cycloheximide was added at a final concentration of 0.1 mg/ml, and eggs were crushed by centrifuging at 13,500 x g in an SW55Ti rotor (Beckman) for 15 min at 4 °C (crushing spin). The cytoplasmic fraction was collected and supplemented with 0.25 mg/ml cycloheximide, and then cleared by re-centrifuging as above. The cleared cytoplasmic fraction (referred to as the low speed supernatant: LSS) was supplemented with 10 µg/ml each of cytochalasin B, aprotinin, leupeptin, and chymostatin and used immediately for experiments. The PEG-M fraction containing MCM2–7 was prepared by differential polyethylene glycol precipitation of LSS, as described previously (49, 50). Immunodepletion of ORC in the egg extracts was achieved by treating the extracts twice (30 min each) with Protein A-Sepharose (Amersham Biosciences) coupled to anti-ORC1 and anti-ORC2 antibodies.

Polyhistidine-tagged, N-terminal-truncated Xenopus geminin (4) was bacterially expressed and purified using nickel-charged Chelating Sepharose column chromatography (Amersham Biosciences). N-terminal-polyhistidine-tagged Xenopus MCM2 was purified as described elsewhere (51). Affinity purification of Xenopus MCM2–7 proteins from the egg extracts was carried out using anti-MCM2 antibodies, essentially as described previously (52, 53). In brief, anti-MCM2 antibodies coupled to Protein A-Sepharose beads were incubated with the egg extracts (LSS) for 1 h at 4 °C.The beads were washed four times with LFB1 buffer (40 mM HEPES-KOH (pH 8.0), 20 mM potassium phosphate (pH 8.0), 2 mM MgCl2, 1 mM EGTA, and 10% sucrose) (49) containing 50 mM KCl but without dithiothreitol, and then MCM proteins were eluted from the beads with LFB1 containing 600 mM KCl (without dithiothreitol) at 4 °C and dialyzed against LFB3 (20 mM HEPES-KOH (pH 8.0), 2 mM dithiothreitol, and 10% sucrose) (49) containing 10 mM KCl. Western blotting showed that the eluate contained all six subunits of MCM2–7 but a relatively low amount of MCM2 was recovered (data not shown).

Formation and Analysis of the Pre-RC—Special care is required to avoid loss of plasmid beads during the experimental procedure. In particular, protein-bound plasmid beads such as those formed after incubation with egg extracts should not be mixed or withdrawn using a micropipette tip. Typically, 40 µg of plasmid beads (equivalent to ~240 ng of plasmid in 4 µl) were withdrawn from the stock using a wide-bore tip and directly mixed with 100 µl of ice-cold XB supplemented with 0.002% Nonidet P-40 in a standard 1.5-ml tube. After brief gentle agitation, the beads were separated using a magnet. The beads were then mixed with 25 µl of egg extracts supplemented with 0.002% Nonidet P-40 (54) and an ATP-regenerating system (20 mM creatine phosphate, 2 mM ATP, and 6.3 µg/ml creatine phosphokinase). The reaction mixture was immediately agitated to disperse the beads uniformly by gently tapping a tube (avoid foaming) and incubated for the indicated times at 23 °C with occasional gentle agitation. After incubation, the reaction mixture was diluted with 150 µl of ice-cold XB supplemented with 0.002% Nonidet P-40 and gently mixed by tapping a tube. The beads were immediately separated on ice using a magnet and as much solution as possible was then removed. The beads were washed three times with 100 µl each of ice-cold XB with 0.002% Nonidet P-40. For Western blot analysis of proteins, the beads were resuspended in SDS-sample buffer and treated at 100 °C for 3 min prior to SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane and incubated with the appropriate antibodies. Detection for Western blotting was done with a Chemi-Lumi One detection kit (Nacalai Tesque, Inc., Kyoto, Japan).


Figure 1
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  		FIGURE 1.
The effect of geminin on pre-RC formation on plasmid beads in Xenopus egg extracts. A, paramagnetic beads coupled with plasmid pG5{lambda}6.6 (lanes 1–6), pKS-EX (lanes 7–12), pBlue{lambda}2.0 (lanes 13–18), or pBluescript (lanes 19–24) were incubated for 30 min in LSS supplemented with increasing amounts of geminin. The proteins bound to the beads were detected by Western blotting with the antibodies as indicated. B, pre-RC formation on plasmid pG5{lambda}6.6 (lanes 1–6), pKS{lambda}7.2 (lanes 7–12), and pEX{lambda}6.6 (lanes 13–18) were analyzed as in A.

 
For quantifying the amounts of ORC1, ORC2, CDC6, and MCM2, each recombinant protein was expressed and purified using bacterial expression vectors for ORC1 (55) and ORC2 (this study) and recombinant baculoviruses for CDC6 (56) and MCM2 (51). Concentrations of the purified proteins were determined by SDS-PAGE/Coomassie Brilliant Blue staining using bovine serum albumin as a standard, and each recombinant protein was then used as a standard in quantitative Western blot analyses of the proteins bound to plasmid beads. The analysis of the proteins bound on sperm chromatin was performed with 2000 demembranated Xenopus sperm nuclei per microliter of egg extracts as described previously (51).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We have recently developed conditions for analyses of both pre-RC formation and DNA replication on circular plasmids in Xenopus egg extracts. In brief, a biotinylated circular plasmid is coupled to streptavidin-paramagnetic beads and used as a template for DNA replication in the egg extracts. We have confirmed that the pre-RC is formed on the circular plasmid in an ORC- and ATP-dependent manner and that DNA synthesis occurs in a pre-RC-dependent manner following nuclear formation.3 To gain further insights into the mechanism of metazoan pre-RC formation, we analyzed pre-RC formation in Xenopus egg extracts using this newly developed approach.

Egg extracts were first mixed with increasing amounts of geminin and tested for pre-RC formation on various kinds of plasmids. On 11-kb plasmid (pG5{lambda}6.6)-coupled beads, loading of MCM3 and MCM6 was inhibited by geminin at 0.5 µg/ml or greater (Fig. 1A, lanes 3–6); these geminin inhibitory concentrations are in agreement with previous results on a sperm chromatin template (4). In contrast, binding of ORC1, ORC2, CDC6, and CDT1 to the plasmid beads was significantly increased at the same geminin concentrations, compared with that in the absence of geminin or at geminin concentrations below 0.3 µg/ml, at which MCM is loaded onto DNA (Fig. 1A, lanes 1–6). None of the proteins bound to beads without coupled plasmids (see Fig. 2C). It should be noted that DNA binding of ORC, CDC6, and CDT1 increased relatively sharply, not gradually, as the geminin concentration increased (Fig. 1A).

With the pKS-EX (5 kb), pBlue{lambda}2.0 (5 kb), and pBluescript (3 kb) plasmids, we unexpectedly found that MCM3 and MCM6 bound to the plasmids even in the presence of geminin concentrations of >0.5 µg/ml (Fig. 1, lanes 7–24). The amount of MCM bound to these plasmid beads did not significantly change in the absence and presence of geminin. In contrast, DNA binding of ORC, CDC6, and CDT1 was increased at higher geminin concentrations for all three plasmids (Fig. 1A), as illustrated for the pG5{lambda}6.6 plasmid (see lanes 3–6). Because MCM binding to plasmid at higher geminin concentrations was also observed with other 10-kb and 12-kb plasmids (Fig. 1B), the plasmid size itself does not seem to be important. We are currently investigating why the 11-kb plasmid pG5{lambda}6.6 behaves differently from the other plasmids with respect to MCM binding in the presence of geminin.


Figure 2
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  		FIGURE 2.
MCM binds to plasmid beads even at higher geminin concentrations, but the binding is unstable and distinct from that in the pre-RC. A, pBluescript-coupled beads were incubated in LSS supplemented with increasing amounts of geminin, and the beads were then washed with the buffer supplemented with (lanes 7–12) or without 0.25% Triton (lanes 1–6). B, after incubation in LSS supplemented with or without 2 µg/ml geminin as in A, the beads were washed at 0 °C (lanes 1, 2, 5, and 6) or at room temperature (lanes 3, 4, 7, and 8), using the buffer supplemented with (lanes 5–8) or without Triton (lanes 1–4). C, pBluescript-coupled beads (lanes 3–8) or beads alone (lanes 1 and 2) were incubated in the ORC-depleted (lanes 3 and 4) or mock-depleted LSS (lanes 1, 2, and 5–8) in the presence or absence of 2 µg/ml geminin, and the beads were then washed with the buffer supplemented with or without Triton at 0 °C as indicated. After washing, the proteins bound to the beads were detected by Western blotting with the antibodies as indicated.

 
MCM binding to plasmids at higher geminin concentrations (0.8 µg/ml or higher in Fig. 2A) was specifically disrupted by washing the beads with 0.25% Triton after incubation in egg extracts (Fig. 2A, lanes 10–12), but MCM loaded at lower geminin concentrations (0.4 µg/ml or lower in Fig. 2A, lanes 7–9) was resistant to Triton treatment. MCM binding at higher geminin concentrations was also disrupted by washing with standard buffer (without Triton) at room temperature, instead of using ice-chilled buffer (Fig. 2B, lane 4), whereas MCM proteins in the pre-RC remained bound to the plasmid beads after washing at room temperature (lane 3). In contrast to the MCM proteins, neither Triton treatment nor washing at room temperature disrupted the increased binding of ORC, CDC6, and CDT1 at higher geminin concentrations (Fig. 2B, lanes 2, 4, 6, and 8). MCM binding to plasmid beads was not detected when ORC or DNA was absent (Fig. 2C, lanes 1–4) or when the incubation was carried out at 0 °C instead of 23 °C (see Fig. 4A). Thus, this weak MCM binding to the plasmid at higher geminin concentrations is not due to nonspecific binding. These results suggest that, even in the presence of an inhibitory amount of geminin, MCM2–7 can interact with a structure that contains at least ORC, CDC6, CDT1, and DNA, but this interaction is unstable and distinct from that in pre-RC formation.

The data above indicate that the protein-DNA complex containing ORC, CDC6, and CDT1 changes dynamically upon stable loading of MCM2–7 onto DNA. To understand this change in more detail, the amount of each initiation protein bound to the plasmid was determined by quantitative Western blotting (Fig. 3). This analysis showed that ~10–20 MCM2 molecules (probably as an MCM2–7 complex) are loaded onto each plasmid molecule after incubation in the absence of geminin, whereas only one or a few molecules of ORC and CDC6 remain bound to the plasmid (Table 1). This MCM:ORC molar ratio is in agreement with previous estimates of the ratio (~20:1) of these proteins bound to sperm chromatin (45) and to linear DNA-coupled beads (46). Thus, the pre-RC is probably formed on circular plasmid-coupled beads in egg extracts in a regulated manner similar to that controlling pre-RC formation on sperm chromatin.


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  		TABLE 1
Quantitation of the proteins bound to plasmid beads during incubation in LSS

The plasmid beads were incubated in LSS for 30 min under the conditions as indicated, and the amounts of the proteins bound to the beads were determined by quantitative Western blotting as shown in Fig. 3. The calculated number of the protein molecules bound to one plasmid molecule is shown. The typical results of four independent experiments are shown.

 


Figure 3
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  		FIGURE 3.
The quantitative Western blot analysis of the proteins bound to plasmid beads. The examples of Western blots for quantitation of the proteins bound to plasmid beads are shown. pKS-EX-coupled beads were incubated in LSS supplemented with or without 2 µg/ml geminin (A) or incubated in LSS at 23 °C or 0 °C (B) for 30 min. After incubation, the beads were washed, and the proteins bound to the beads (left two lanes, equivalent to 4 µl(A) or 1 µl(B) of the beads) were analyzed along with the standards of the recombinant proteins.

 
Interestingly, more than 10 molecules each of ORC and CDC6 were bound to each plasmid at geminin concentrations at which stable MCM loading (pre-RC formation) was inhibited (Table 1). In addition, almost the same amount of MCM interacted with the plasmid (except for pG5{lambda}6.6) regardless of the presence or absence of geminin (Fig. 1, also see Table 1). Thus, ORC, CDC6, and the MCM2–7 complex bind to each plasmid at an approximate molar ratio of 1:1:1 in the presence of inhibitory amounts of geminin, although MCM binding is unstable under these conditions.

The data above imply that DNA binding of ORC and CDC6 may also change during the process of pre-RC formation in the absence of geminin. Related to this, it has been reported that the stability of ORC-chromatin binding changes before and after pre-RC formation (57). Thus, we next analyzed DNA binding of the pre-RC components to plasmids during incubation in egg extracts in the absence of geminin. Under these conditions at 0 °C, plasmid binding of ORC, CDC6, and CDT1 markedly increased (Fig. 4A, lanes 3, 7, and 11). Because prior to the start of incubation at 23 °C the reaction ingredients were mixed in a tube kept on ice, these results indicate that ORC, CDC6, and CDT1 had already accumulated on the plasmid-coupled beads before incubation at 23 °C. Increased binding at 0 °C was not seen in ORC-depleted extracts (Fig. 4B, lane 6), indicating that increased binding of CDC6 and CDT1 at 0 °C is indeed ORC-dependent. MCM loading was not detectable at 0 °C in the presence or absence of geminin (Fig. 4A). More importantly, the levels of ORC, CDC6, and CDT1 bound at 0 °C were similar to those previously seen after incubation at 23 °C in the presence of an inhibitory amount of geminin (Fig. 4A and Table 1). Thus, it is likely that a similar ORC-CDC6-CDT1 complex is formed and stabilized on DNA under conditions where MCM loading is inhibited by incubation at 0 °C or by an inhibitory geminin concentration at 23 °C. Increased binding of ORC, CDC6, and CDT1 to chromatin without MCM loading also occurred with sperm chromatin at 0 °C (Fig. 4C, lane 3), indicating that this binding is not specific for plasmid-coupled beads. Likewise, increased binding to sperm chromatin also occurred when an inhibitory amount of geminin was added (Fig. 4C, lane 2, also see the Ref. 4). Therefore, increased DNA/chromatin binding of ORC, CDC6, and CDT1 appears to be a general consequence of inhibition of MCM loading. Consistent with this, it has been shown that ORC-chromatin binding is significantly stabilized in MCM-depleted egg extracts (57).


Figure 4
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  		FIGURE 4.
The binding of ORC, CDC6, and CDT1 to plasmid beads is increased during incubation at 0 °C. A, the paramagnetic beads coupled with pG5{lambda}6.6 (lanes 1–4), pKS-EX (lanes 5–8), or pBluescript (lanes 9–12) were incubated for 30 min in LSS supplemented with or without 2 µg/ml geminin at 23 °C or 0 °C as indicated. B, pBluescript-coupled beads were incubated in the mock-depleted (lanes 3 and 5) or the ORC-depleted LSS (lanes 4 and 6) at 23 °C or 0 °C for 30 min, in the presence or absence of 2 µg/ml geminin as indicated. The proteins bound the beads in A and B were then analyzed by Western blotting with the appropriate antibodies as indicated. Lanes 1 and 2 in B represent 1 of 17 of the depleted LSS used for the binding reaction. C, demembranated Xenopus sperm nuclei were incubated for 30 min at 23 °C or 0 °C in LSS supplemented with or without 2 µg/ml geminin, and the proteins bound to chromatin were analyzed as in A and B.

 
A time-course analysis showed that ORC, CDC6, and CDT1 accumulated on plasmid beads as soon as 1 min after incubation at 0 °C (Fig. 5A, lane 5). Increased levels of these bound proteins were then retained for at least 30 min (Fig. 5A, lanes 5–7). Binding of these proteins was not detectable without DNA (Fig. 5A, lanes 2–4), indicating that binding at 0 °C is DNA-dependent. More interestingly, upon shifting the temperature to 23 °C after a 30-min incubation at 0 °C, ORC1, CDC6, and CDT1 all were gradually displaced from DNA; in particular, CDC6 was displaced most rapidly, with a significant portion of CDC6 being displaced 1 min after the shift to 23 °C (Fig. 5B, lane 3). Conversely, MCM3 loading onto DNA began immediately after the shift to 23 °C. The level of loaded MCM3 reached a plateau after 15 min at 23 °C, whereas bound CDC6 was undetectable at this time point (Fig. 5B, lane 4). These results indicate that DNA binding of ORC, CDC6, and CDT1 changes dynamically during the process of de novo pre-RC formation in the absence of geminin.

Based on the above observations, we hypothesized that the ORC-CDC6-CDT1 complex formed on DNA at 0 °C may represent an intermediate that acquires an ability to directly load MCM2–7 onto DNA. To investigate this further, plasmid beads were first incubated with egg extracts at 0 °C to allow formation of the ORC-CDC6-CDT1 complex on DNA. After this, the beads were isolated using a magnet, washed to remove unbound proteins, and then subjected to a second incubation with the PEG-M fraction, which contains MCM2–7 but not ORC1, CDC6, or CDT1 (Refs. 49 and 50 and data not shown). As shown in Fig. 6A, MCM was loaded onto the plasmid during the second incubation with PEG-M in a time-dependent manner (lanes 7–10), suggesting that the ORC-CDC6-CDT1 complex formed on DNA is indeed able to load MCM2–7 onto the DNA. To exclude the possibility that some unknown factor(s) in the PEG-M fraction may function in MCM loading, along with the ORC-CDC6-CDT1 complex, the same MCM loading assay was carried out using affinity-purified MCM proteins, instead of the PEG-M fraction. As shown in Fig. 6B, MCM was also loaded onto DNA under these conditions (lane 3). Geminin inhibited MCM loading in these experiments, as expected (Fig. 6B, lane 5). Moreover, MCM loading required the presence of ATP in the second incubation (Fig. 6B, lane 4) and seemed to require the entire MCM2–7 complex (lane 6), because MCM loading was suppressed when the second incubation mixture was not supplemented with recombinant MCM2 (note that the affinity-purified MCM contained a relatively low amount of MCM2 because of the use of an anti-MCM2 antibody-column for purification; see "Experimental Procedures"). Although many bands other than those for MCM proteins were detected in the affinity-purified MCM fraction by SDS-PAGE and silver staining (data not shown), these results are consistent with the hypothesis that the ORC-CDC6-CDT1-DNA complex formed at 0 °C is directly involved in MCM loading. However, it should be noted that, in contrast with previous results (Fig. 5B), ORC, CDC6, and CDT1 were not significantly displaced from DNA in these stepwise reactions, even after MCM loading (Fig. 6).


Figure 5
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  		FIGURE 5.
ORC, CDC6, and CDT1 are gradually displaced from DNA upon MCM loading. A, pBluescript-coupled beads (lanes 5–7) or the beads alone (lanes 2–4) were incubated in LSS at 0 °C for the indicated times. B, pBluescript-coupled beads were incubated as in A for 30 min (lane 2), the temperature was then shifted to 23 °C, and the reaction mixtures were further incubated for the indicated times (lanes 3–5). The proteins bound to the beads in A and B were analyzed by Western blotting. Lane 1, 1 of 25 LSS used for the reaction.

 


Figure 6
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  		FIGURE 6.
The ORC-CDC6-CDT1 complex formed on plasmid beads is capable of loading MCM proteins onto plasmid. A, pG5{lambda}6.6-coupled beads were incubated in LSS for 30 min at 0 °C, washed, and then subjected to a second incubation with PEG-M (lanes 7–10) or the control buffer (lanes 3–6) at 23 °C for the indicated times. B, incubation in LSS at 0 °C was performed as in A, and the plasmid beads were washed, and then subjected to a second incubation for 30 min at 23 °C with purified MCM proteins supplemented with purified recombinant MCM2 (lane 3). The same second incubation was also performed without ATP (lane 4), in the presence of geminin (lane 5), or without a supplement of recombinant MCM2 (lane 6). Lanes 1 and 2 represent the incubation in LSS for 30 min at 23 °C and 0 °C, respectively. The proteins bound to the beads were analyzed by Western blotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is well known that the number of MCM2–7 complexes loaded onto chromatin is much larger than the number of ORC molecules bound to chromatin (the ORC number presumably reflects the number of replication origins) (45, 46). MCM complexes are also known to be widely distributed over chromatin before the onset of DNA replication in the nuclei of mammalian cells and in reconstituted nuclei in Xenopus egg extracts (41–43). The data presented here have important implications for the mechanism of MCM loading onto chromatin in metazoans.

DNA binding of ORC, CDC6, and CDT1 was significantly stabilized by either the presence of geminin or incubation at low temperature, conditions under which MCM loading was inhibited. This stabilization is consistent with the previous observation that ORC and CDC6 bind more tightly to sperm chromatin in MCM-depleted extracts (57). Quantitative analysis showed that ~10–20 molecules each of ORC and CDC6 are bound to each plasmid (stage I in Fig. 7). Cooperative binding of S. cerevisiae ORC and CDC6 to the origin has also been recently reported at this stage, and formation of a ring-shape ORC-CDC6 complex consisting of one ORC and one CDC6 molecule has been proposed (22). Our estimate of an approximate ORC:CDC6 molar ratio of 1:1 without MCM loading is consistent with this model. However, unlike the S. cerevisiae ORC, multiple Xenopus ORC-CDC6 complexes (more than 10 molecules) may bind randomly to the plasmid (stage I in model A), because Xenopus ORC does not have sequence-specific DNA binding activity. Alternatively, multiple ORC-CDC6 complexes may aggregate at a limited number of sites on the plasmid (stage I in model B). Curiously, the plasmid size does not seem to affect the number of ORC-CDC6 complexes bound to the plasmid significantly; ~10–20 ORC-CDC6 complexes were bound to the 5- and 11-kb plasmids in the presence of geminin or at decreased temperature (Table 1). Consistent with this observation, a relatively constant amount of ORC was shown to bind to linear DNA in Xenopus egg extracts, irrespective of the length of DNA (longer than 0.35 kb) (46). Thus, ORC-CDC6 binding to DNA in metazoans might also be regulated by unknown mechanisms. We have not estimated the number of bound CDT1 molecules, but it is likely that CDT1 binds stoichiometrically to the ORC-CDC6-DNA complex, because CDT1 was shown to interact with CDC6 and DNA in addition to MCM proteins (58, 59).


Figure 7
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  		FIGURE 7.
The model for multiple MCM loading on plasmid in Xenopus egg extracts. At first, multiple ORC-CDC6-CDT1 complexes may be formed randomly on plasmid (model A) or aggregate at a limited number of sites on plasmid (model B) (stage I). This state can be stabilized if MCM loading is prevented (e.g. at decreased temperature). In the presence of geminin, an MCM2–7 complex may interact with an individual ORC-CDC6-CDT1 complex (stage II), but this interaction is unstable. Upon loading of MCM onto DNA by ORC-CDC6-CDT1 complexes, ORC, CDC6, and CDT1 are displaced from DNA, leaving multiple MCM complexes and only one or a few ORC molecules on DNA (stage III). Note that CDT1 is omitted in this figure. See "Discussion" for the detail.

 
In the presence of an inhibitory amount of geminin at 23 °C, the process appears to advance one step further toward pre-RC formation; that is, MCM proteins may associate with the ORC-CDC6-CDT1 complex on DNA, although this association is very weak (stage II in Fig. 7). MCM association with the ORC-CDC6-CDT1 complex in the presence of geminin is in agreement with the domain structure of CDT1, in which the domains for geminin and MCM binding are separable (59). Our estimate suggests that MCM2–7 binds to the ORC-CDC6-CDT1 complex at a molar ratio of 1:1 in the presence of geminin. Moreover, the number of these weakly associated MCM complexes was roughly the same as the number remaining on DNA after pre-RC formation. Although it is unclear if this MCM-associated complex is an intermediate in pre-RC formation in vivo, stoichiometric association of MCM with the ORC-CDC6-CDT1 complex may reflect a specific state during pre-RC formation. Taken together with our observations that a similar ORC-CDC6-CDT1 complex is formed during incubation at 0 °C in the absence of geminin and that the isolated ORC-CDC6-CDT1-DNA complex can directly load the MCM complex onto DNA, it seems likely that an individual ORC-CDC6 complex interacts with an MCM2–7 complex and loads it onto DNA, leading to loading of multiple MCM complexes onto each plasmid. In addition, the number of ORC-CDC6-CDT1 complexes formed before MCM loading was estimated to be similar to the number of MCM2–7 complexes present after MCM loading, suggesting that each ORC-CDC6-CDT1 complex may load only one or a few MCM2–7 complexes. It was not possible to determine the number of loaded MCM complexes more precisely, due to the limited accuracy of the immunoblotting procedure used in this study, and determination of this number will require experiments with highly purified proteins.

Upon loading of the MCM complex onto DNA, ORC, CDC6, and CDT1 were displaced, leaving only one or a few ORC molecules and ~10–20 MCM complexes on DNA (an ORC:MCM2–7 complex ratio of ~1:10–20; stage III in Fig. 7). A previous estimation of the ORC: MCM ratio (~1:20) in Xenopus DNA replication (45, 46) may reflect this stage. Destabilization of chromatin-bound ORC and CDC6 is well known during DNA replication in Xenopus egg extracts (56, 60), and our time-course analysis also showed that CDC6, in particular, is displaced from DNA relatively quickly (Fig. 5B). Although the possibility cannot be formally excluded that only a small population of the ORC-CDC6-CDT1 complexes that assembled on DNA at 0 °C remain on the DNA to load multiple MCM2–7 complexes repeatedly, the relative correlation between MCM loading and the displacement of ORC, CDC6, and CDT1 (Fig. 5B) does not fit with this possibility. Rather, it is likely that the ORC-CDC6-CDT1 complex is displaced from DNA once it has loaded one or a few MCM complexes.

Because CDC6 and the ORC1–5 subunits are AAA+-related proteins, it is possible that conformational changes induced by ATP binding and hydrolysis may occur in ORC and/or CDC6 and decrease the ORC-CDC6 interaction and/or the affinity of ORC or CDC6 for DNA. These changes may also be coupled with MCM loading. However, MCM loading may not be sufficient for displacing ORC, CDC6, and CDT1 from DNA, because the stepwise MCM loading shown in Fig. 6 did not lead to displacement of these proteins. Hence, some other unknown mechanism may also be involved in this displacement. We noticed that a small population of ORC molecules (about one ORC per plasmid) consistently remained bound to the DNA after pre-RC formation. It is well established that ORC is dispensable for the steps in DNA replication subsequent to pre-RC formation (60), but it is possible that remaining ORC molecules may have other roles in DNA replication, such as in selecting the MCM complex to be activated initially, or in other activities taking place on chromatin.

A recent FLIP (fluorescence loss in photobleaching) analysis of ORC-chromatin binding in mammalian cells revealed that ORC rapidly associates with and dissociates from chromatin throughout the interphase of the cell cycle (61), suggesting that DNA binding of metazoan ORC is much more dynamic than previously thought. Thus, it is possible that ORC repeatedly binds at multiple sites on chromatin and loads one or a few MCM complexes per ORC-chromatin binding event as long as the nuclear environment allows pre-RC formation; i.e. a low geminin concentration and the presence of CDT1 (2). These dynamic ORC binding and MCM loading events may account for the wide distribution of the MCM complex over chromatin before the onset of DNA replication, as seen in mammalian cell nuclei and in synthetic nuclei in Xenopus egg extracts. We believe, therefore, that the data in this study provide important information on the mechanism of pre-RC formation and in vivo ORC functions in metazoans. Further investigation of the mechanism and regulation of ORC-DNA binding and MCM loading will lead to an understanding of how replication origins are established on chromosomal DNA in metazoans.

It has been known that Xenopus egg extracts contain the activities for DNA synthesis-dependent and -independent chromatin assembly (62). We confirmed that, in parallel with pre-RC formation, nucleosomes are assembled on plasmid-coupled beads in LSS.4 Thus, one can imagine that chromatin structure may affect more or less DNA binding of ORC and the subsequent MCM loading. Although it has not been known exactly how chromatin structure affects these reactions, persistent binding of ORC, CDC6, and CDT1 after MCM loading shown in Fig. 6 may result from chromatin structure formed during the stepwise reaction, which might be distinct from that formed in a standard reaction in LSS. In addition to chromatin formation, some protein-DNA complexes may be formed on a plasmid in a sequence-specific manner and affect pre-RC formation directly or indirectly. With respect to this, we speculate that some protein-DNA complexes might be formed specifically on plasmid pG5{lambda}6.6, leading to suppression of MCM binding to pG5{lambda}6.6-coupled beads in the presence of geminin as shown in Fig. 1. Thus, because multiple events, including pre-RC formation and nucleosome assembly, occur simultaneously on a plasmid in the crude egg extracts, it will be necessary to reconstitute these events with purified components to know precisely how pre-RC formation is affected by DNA sequences, DNA-binding proteins, or chromatin structure.

A ring-shaped structure of the S. cerevisiae ORC-CDC6 complex has recently been described, with dimensions similar to that of the ring-shaped MCM complex structure (22). This may imply that an ORC-CDC6 complex directly interacts with an MCM ring and opens it for loading onto DNA, in a manner reminiscent of loading of a DNA polymerase clamp (proliferating cell nuclear antigen) by a clamp loader (replication factor C) (63, 64). Our data also support the direct involvement of the ORC-CDC6 complex in MCM loading. Taken together with the previous reconstitution of the Xenopus pre-RC from purified proteins (65), these results suggest that ORC, CDC6, and CDT1 are a minimum set of proteins that are directly involved in MCM loading. It is of note that there are structural and functional similarities between the eukaryotic ORC-CDC6 complex and MCM2–7 helicase and the Escherichia coli DnaA protein and DnaB hexameric DNA helicase, respectively (22, 63, 66, 67). However, in the case of E. coli DNA replication, another AAA+-family protein, DnaC, functions as a loader of DnaB helicase (63). Therefore, it will be intriguing to determine whether the ORC-CDC6 complex has dual roles reflecting those of DnaA and DnaC during MCM loading, or whether another protein, such as CDT1 and/or a subset of MCM proteins such as MCM3 and -5, have a role similar to that of DnaC.


   FOOTNOTES
 
* This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas (17013054 to S. W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Yamada Science Foundation (to S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel./Fax: 81-(0)6-6879-4660; E-mail: swaga{at}fbs.osaka-u.ac.jp.

2 The abbreviations used are: ORC, origin recognition complex; MCM, mini-chromosome maintenance; pre-RC, pre-replicative complex; LSS, low speed supernatant. Back

3 A. Zembutsu and S. Waga, manuscript in preparation. Back

4 A. Zembutsu and S. Waga, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank T. Seki for valuable technical advice, A. Furukohri for purification of recombinant MCM2 protein, H. Takisawa, J. Walter, and S. Tada for providing the antibodies, and P. Carpenter, M. Shirakata, T. McGarry, and Y. Ohkuma for providing the plasmids. We also thank A. Sugino, H. Shinagawa, and F. Hanaoka for encouraging us throughout this work.



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