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Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G1 transition in mammals

2000; Springer Nature; Volume: 19; Issue: 11 Linguagem: Inglês

10.1093/emboj/19.11.2728

1460-2075

Darren A. Natale,

Microtubule and mitosis dynamics

Article1 June 2000free access Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M–G1 transition in mammals Darren A. Natale Darren A. Natale Present address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20894 USA Search for more papers by this author Cong-Jun Li Cong-Jun Li National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA Search for more papers by this author Wei-Hsin Sun Wei-Hsin Sun National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA Search for more papers by this author Melvin L. DePamphilis Corresponding Author Melvin L. DePamphilis National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA Search for more papers by this author Darren A. Natale Darren A. Natale Present address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20894 USA Search for more papers by this author Cong-Jun Li Cong-Jun Li National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA Search for more papers by this author Wei-Hsin Sun Wei-Hsin Sun National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA Search for more papers by this author Melvin L. DePamphilis Corresponding Author Melvin L. DePamphilis National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA Search for more papers by this author Author Information Darren A. Natale2, Cong-Jun Li1, Wei-Hsin Sun1 and Melvin L. DePamphilis 1 1National Institute of Child Health and Human Development, Building 6, Room 3A02, National Institutes of Health, Bethesda, MD, 20892-2753 USA 2Present address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20894 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2728-2738https://doi.org/10.1093/emboj/19.11.2728 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G1 phase, and reappeared as cells progressed through G1 phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G1 phase, but became stably bound during mid-G1 phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed. Introduction How do mammalian cells initiate DNA replication at specific sites along their chromosomes? During the past few years, it has become clear that most, if not all, of the proteins used to initiate DNA replication in the budding yeast, Saccharomyces cerevisiae, are also used to initiate DNA replication in many, perhaps all, eukaryotes. Homologs for many of these proteins have been identified in yeast, fungi, plants, nematodes, frogs, flies and mammals (a current list of the organisms encoding each protein and their literature citations can be found by searching the protein database at http://www.ncbi.nlm.nih.gov). They include the six Orc proteins that comprise the origin recognition complex (ORC), cell division cycle (Cdc) proteins 6 and 45, six mini-chromosome maintenance (Mcm) proteins and the Cdc7 protein kinase and its cofactor Dbf4. Several of these homologs are required for DNA replication. Orc2 and Dbf4 proteins are required in Drosophila (Landis et al., 1997; Landis and Tower, 1999). Orc, Cdc6, Mcm, Cdk2 and Cdk7/Dbf4 proteins are required in Xenopus (Carpenter et al., 1996; Coleman et al., 1996; Romanowski et al., 1996; Rowles et al., 1996; Walter and Newport, 1997; Hua and Newport, 1998). Mcm, Cdc6, Cdk2 and Cdc7/Dbf4 proteins are required in mammals (Todorov et al., 1994; Krude et al., 1997; Yan et al., 1998; Jiang et al., 1999a; Kumagai et al., 1999). Thus, the mechanism for DNA replication is highly conserved among eukaryotes. As with yeast, initiation of DNA replication in the differentiated cells of frogs, flies and mammals also begins predominantly at specific replication origins (DePamphilis, 1999; Phi-van and Stratling, 1999). Initiation of bidirectional DNA replication at these sites depends on both proximal (Handeli et al., 1989; Aladjem et al., 1998; Malott and Leffak, 1999) and distal (Aladjem et al., 1995; Kalejta et al., 1998) cis-acting DNA sequences. In addition, the activity of individual replication origins can depend on nuclear structure, chromatin structure, the ratio of initiation factors to DNA, and DNA methylation [reviewed in DePamphilis (1999) and Rein et al. (1999)]. The combination of genetic and epigenetic parameters can account for the changes that can occur in the number and locations of initiation sites as rapidly cleaving embryos develop into differentiated organisms (Hyrien et al., 1995; Sasaki et al., 1999). One difference that may exist between yeast and metazoan cells is the way in which the ORC interacts with chromatin, a step that is critical to understanding how initiation sites are selected. In yeast, both DNA footprinting (Diffley et al., 1994; Fujita et al., 1998) and immunoprecipitation (Liang and Stillman, 1997) analyses reveal that a complete ORC binds to yeast replication origins immediately after initiation of replication occurs and remains there throughout the cell division cycle. In metazoa, however, the situation is less clear. Some evidence suggests that the ORC dissociates from chromatin during mitosis. G1 phase nuclei from mammalian cells can initiate DNA replication when incubated in a Xenopus egg extract that has been depleted of Xenopus laevis Orc (XlOrc) proteins (Romanowski et al., 1996; Yu et al., 1998), whereas metaphase chromatin from mammalian cells replicates poorly under these conditions (Yu et al., 1998). These data suggest that mammalian Orc proteins are absent from chromatin during metaphase and then rebind at some time before G1 phase begins. Consistent with this conclusion, Orc proteins in activated Xenopus eggs bind to sperm chromatin, whereas Orc proteins in mitotic Xenopus eggs do not (Coleman et al., 1996; Hua and Newport, 1998; Findeisen et al., 1999; Rowles et al., 1999), and both Orc1 and Orc2 proteins are present on chromatin in cultured Xenopus cells during interphase but not during metaphase (Romanowski et al., 1996). However, in Drosophila, Orc2 is present in both interphase and metaphase (Pak et al., 1997), and human Orc2 is present on cells throughout the cell cycle, although metaphase cells per se were not examined (Ritzi et al., 1998). These data appear to contradict the work in Xenopus, suggesting that the behavior of ORC may vary even among metazoa. Therefore, it is difficult to relate the presence of Orc activity to the presence of Orc proteins in metazoa, because different assays for ORC function or Orc proteins have been carried out in different organisms. Moreover, the hypothesis that functional, chromatin-bound ORCs exist on mammalian chromatin throughout G1 phase is difficult to reconcile with the presence of an 'origin decision point' (ODP) (Wu and Gilbert, 1996) in mammalian cells; initiation of DNA replication can be induced by a Xenopus egg extract in all G1 nuclei, but initiation events in late G1 nuclei occur at the same replication origins used in vivo whereas initiation events in early G1 nuclei are randomly distributed along the genome (Wu and Gilbert, 1996; Yu et al., 1998; Li et al., 2000). The work presented here provides a solution to this paradox by showing that the mammalian ORC disassembles, at least in part, during mitosis and then reassembles completely in early G1 nuclei. In Chinese hamster ovary (CHO) cells, CgOrc2 (where Cg indicates Cricetulus griseus, the Chinese hamster) remained stably bound to chromatin throughout the cell cycle, consistent with studies of Drosophila and human cells, but CgOrc1 became unstably bound to chromatin during mitosis, consistent with studies in Xenopus cells. Moreover, the ability to initiate DNA synthesis in hamster cells and the ability to activate specific mammalian replication origins selectively by incubation in Xenopus egg extract were directly related to the stability of CgOrc1 binding to chromatin. These results support the general concept that metazoan ORCs, in contrast to yeast ORCs, dissociate from chromatin during mitosis. In addition, they reveal that mammals selectively destabilize Orc1 protein during the mitotic to G1 transition, thus providing a novel mechanism in mammalian cells for delaying assembly of pre-replication complexes (pre-RCs), consisting of ORC, Cdc6 and one or more copies of Mcm proteins 2–7, until mitosis is complete and a nuclear membrane has reformed. Finally, they strongly suggest that mammalian ORCs are assembled at specific chromosomal sites. Results Functional hamster Orc proteins are absent during mitosis and early G1 phase The presence of functional ORCs on mammalian chromatin was assayed by the ability of an Orc-depleted Xenopus egg extract to initiate DNA replication in hamster nuclei or metaphase chromatin. Previous studies have shown that antibodies against XlOrc1 or XlOrc2 co-precipitate other XlOrc proteins (Romanowski et al., 1996; Carpenter and Dunphy, 1998; Tugal et al., 1998), thus depleting egg extract of most, if not all, of their Orc proteins. In our hands, agarose beads coated with anti-XlOrc2 serum removed ≥98% of the XlOrc2 protein from the depleted extract, while <1% of the XlOrc2 protein was lost from the mock-depleted extract (Li et al., 2000). Depletion of XlOrc1 or XlOrc2 proteins from Xenopus egg extract prevents initiation of DNA replication in sperm chromatin or plasmid DNA substrates (Carpenter et al., 1996; Coleman et al., 1996; Romanowski et al., 1996; Rowles et al., 1996, 1999; Yu et al., 1998), demonstrating that initiation of DNA replication in Xenopus egg extract is dependent on a functional interaction between ORC and chromatin. Similarly, the XlOrc-depleted extracts used here did not support DNA replication in Xenopus sperm chromatin (Figure 1), confirming that XlOrc proteins were functionally, as well as physically, absent. Figure 1.Orc proteins were required to initiate DNA synthesis in hamster chromatin. Xenopus sperm chromatin (1.6 × 105) or nuclei prepared by digitonin lysis (25 000/μl) were tested for their ability to replicate when incubated in complete Xenopus egg extract (+XlOrc2, filled squares), XlOrc2-depleted extract (−XlOrc2, open squares) or mock-depleted extract (open circles) supplemented with [α-32P]dATP and [α-32P]dCTP. The amount of acid-precipitable 32P-labeled DNA was expressed as % DNA synthesis relative to the incorporation observed after 1 h of incubation in complete extract. The mean ± SEM is given for three or more independent experiments in complete or depleted extract. One experiment was performed with mock-depleted extract. The amount of DNA synthesized by 1 h (0.4 pmol dAMP × 10-6/nucleus) was equivalent to 10–15% of the genome replicated, comparable to previous studies (Gilbert et al., 1995; Wu et al., 1997). Download figure Download PowerPoint Hamster cells were synchronized in mitosis and released into G1 phase; metaphase chromatin (0 h) or G1 phase nuclei (1–6 h) were then isolated by permeabilizing the cells with digitonin. When these substrates were incubated in either complete or mock-depleted Xenopus egg extract, the time courses for DNA synthesis (Figure 1) were typical of those reported previously (Gilbert et al., 1995). However, when the same substrates were incubated in XlOrc-depleted extract, a clear transition was observed from dependence to independence of DNA replication on XlOrc proteins (Figure 1). Hamster metaphase chromatin had the same dependence on XlOrc proteins as Xenopus sperm chromatin, and DNA synthesis was not detected during the first hour of incubation in nuclei isolated 1 h after metaphase. Hamster nuclei isolated 1.5 h after metaphase were able to function partially in the absence of Xenopus Orc proteins, while nuclei isolated ≥2 h after metaphase no longer required the presence of XlOrc proteins to initiate DNA synthesis. Therefore, hamster Orc proteins are not functional until 1–2 h after metaphase. Acquisition of site-specific DNA replication in hamster nuclei coincides with independence from XlOrc proteins Previous studies using Xenopus egg extract to initiate DNA replication in hamster G1 phase nuclei revealed that site-specific initiation did not appear until mid-G1 phase (the ODP). Before that point, DNA synthesis appeared 'randomly' distributed throughout the dihydrofolate reductase (DHFR) gene region. After that point, DNA synthesis was largely confined to the 55 kb intergenic region between the DHFR gene and the 2BE2121 gene as a broad peak of DNA synthesis centered at the ori-β/β′ locus. Therefore, we considered the possibility that the ODP coincided with the functional association of hamster Orc proteins with hamster chromatin. In an effort to confirm and refine the ODP, and to relate it to the results reported here, the ODP was mapped using a simplified experimental protocol that allowed Xenopus egg extract to initiate DNA replication selectively at the same primary initiation sites used by CHO cells in vivo, resulting in much sharper peaks of origin activity than previously reported (Li et al., 2000). Hamster G1 phase nuclei were incubated in Xenopus egg extract for 45 min to label the initial burst of newly synthesized DNA with [α-32P]dATP and [α-32P]dCTP, most of which consisted of RNA-primed DNA chains from 0.5 to ∼2 kb in length. Under these conditions, site-specific initiation events were not observed in 1 h G1 nuclei when DNA synthesis was dependent on the presence of XlOrc proteins (Figure 2). However, ori-β was selectively activated in G1 nuclei isolated ≥2 h after release from mitosis, and this activation, like total DNA synthesis (Figure 1), was independent of Xenopus Orc proteins (Li et al., 2000; data not shown). Therefore, the appearance of ori-β activity resulted from activation of hamster pre-RCs by Xenopus egg extract. Figure 2.The ability of Xenopus egg extract to activate ori-β selectively in hamster nuclei increases as hamster cells progress through G1 phase. Nuclei were isolated 1, 2, 3, 4 or 5 h after release of CHOC 400 cells from mitosis and incubated for 45 min in Xenopus egg extract supplemented with [α-32P]dATP and [α-32P]dCTP. The resulting 32P-labeled DNA was hybridized to specific DNA probes, and the results from each time point were then divided by the results from 1 h G1 nuclei. Thus, the data for 1 h G1 nuclei are represented by the horizontal dashed line (open circles), and the increase above this level of DNA synthesis at each subsequent time point by the solid line (filled circles). Download figure Download PowerPoint To determine the time course for the appearance of hamster pre-RCs at ori-β, nuclei were isolated at various times after metaphase and incubated in Xenopus egg extract. The newly synthesized 32P-labeled DNA present after 45 min was then hybridized to specific DNA sequences, and the counts/base pair of 32P-labeled DNA that hybridized to each probe was divided by the corresponding value obtained with 1 h G1 nuclei (Figure 2). This ratio provided a quantitative assessment of the extent to which ori-β could be selectively activated as cells progressed through G1 phase. Pre-RCs were first detected at ori-β 1.5–2 h after metaphase, and their assembly within the cell population was complete 4–5 h after metaphase, or ∼2 h before the onset of S phase (Figure 3). This result was similar to that of Wu and Gilbert (1996) except that the ODP determined here occurred ∼1 h earlier. Figure 3.Non-specific initiation of DNA replication in hamster G1 nuclei was dependent on Xenopus Orc proteins, but site-specific initiation (e.g. ori-β) was not. % XlOrc dependence (filled diamonds) is the % DNA synthesis observed after CHOC 400 nuclei had been incubated for 1 h in Xenopus egg extract (Figure 1). % Ori-β activity (filled circles) is the height of the peak at ori-β observed at the indicated time relative to the height observed 4 h after metaphase (Figure 2). % S-phase cells (filled triangles) is the percentage of cells labeled with BrdU in vivo. DNA synthesis (open triangles) is the amount of [3H]thymidine-labeled cells incorporated in vivo (104 c.p.m./105 cells). Download figure Download PowerPoint The transition from XlOrc dependence to XlOrc independence of DNA replication in hamster chromatin coincided with the transition from non-specific ('random') to site-specific initiation of DNA replication in hamster chromatin (Figure 3). Before the ODP, DNA replication in hamster chromatin depended on the presence of XlOrc proteins and was initiated 'randomly' throughout the chromatin. After the ODP, DNA replication in hamster chromatin was independent of XlOrc proteins and occurred preferentially at specific sites such as ori-β. Therefore, the ability of Xenopus egg extract to activate selectively specific replication origins in hamster nuclei in vitro required prior assembly of hamster pre-RCs in vivo. CgOrc1 and CgOrc2 levels are constant during mitosis and G1 phase The results summarized in Figure 3 suggested that CgOrc proteins were not functionally associated with hamster chromatin until mid-G1 phase. To monitor the presence of these proteins, the CgORC1 and CgORC2 genes were cloned and expressed, and antibodies were produced against the expressed proteins. CgORC1 encodes an 850 amino acid polypeptide (CgOrc1) with a predicted mol. wt of 96 kDa. CgORC2 encodes a 576 amino acid polypeptide (CgOrc2) with a predicted mol. wt of 66 kDa. Hamster amino acid sequences are closely related to their human and mouse homologs (77% similar for Orc1; 88% similar for Orc2), particularly in their C-terminal ends [93% for Orc1 (CgOrc1 amino acids 484–850); 97% for Orc2 (CgOrc2 amino acids 297–576); Figure 4]. The antisera readily detected proteins of the expected molecular weights in hamster cells that co-migrated during gel electrophoresis with samples of the expressed proteins (Figure 5A). Figure 4.Comparison of the predicted amino acid sequences of mammalian Orc1 (A) and Orc2 (B) proteins. DDBJ/EMBL/GenBank identification numbers for Orc1 are: Chinese hamster (Cg), AF254572; mouse (Mm), 4034785; and human (Hs), 4758850; those for Orc2 are: Chinese hamster, AF254573; mouse, 2498710; and human, 5453830. Identical amino acids are in black. Similar amino acids are in gray. Download figure Download PowerPoint Figure 5.Total CgOrc1 and CgOrc2 protein levels remained constant during mitosis and G1 phase. CHOC 400 cells were arrested in mitosis and then released into G1 phase. At the indicated time, cells were lysed with SDS. One aliquot was fractionated by electrophoresis in a 4–14% polyacrylamide gradient gel and assayed for Orc1 while another aliquot was fractionated and assayed for Orc2. (A) Antibody specificity was confirmed by immunoblotting samples of G1 phase cell extracts run in parallel with purified CgOrc1 or CgOrc2 protein and molecular weight markers. (B) Amounts of Orc1 and Orc2 at each time point were determined by subjecting the top portion of each gel to immunoblotting while the bottom portion of the gel was stained to detect histones. Individual bands were quantified by densitometry. Relative amounts of Orc protein were determined from the Orc:histone ratio in each lane. Total amounts of Orc protein were estimated from the intensities of samples of purified Orc proteins run in parallel. (C) At the indicated time, cells were permeabilized with digitonin and processed as above except that samples were fractionated in a 12% polyacrylamide gel and the entire gel was subjected to immunoblotting simultaneously with the indicated anti-CgOrc IgG and anti-actin IgM. (D) The mean ± SEM at each time point was determined for six experiments. DNA synthesis is the amount of [3H]thymidine-labeled cells incorporated in vivo (DNA synthesis, 103 c.p.m./105 cells, open triangles) and the fraction of cells labeled with BrdU in vivo (filled triangles, plotted on the left y-axis). Download figure Download PowerPoint To quantify the relative concentrations of CgOrc1 and CgOrc2 during mitosis and G1 phase, hamster cells were isolated at different times after their release from metaphase and lysed with sodium dodecyl sulfate (SDS). Separate aliquots of the lysate were then fractionated by gel electrophoresis and subjected to immunoblotting with CgOrc1 or CgOrc2 antiserum. The bottom portion of each gel was stained separately to detect histones and the amount of Orc protein in each lane was normalized to the amount of histone present. Samples of purified CgOrc1 or CgOrc2 protein of known concentrations were included during the gel fractionation step in order to identify the correct protein and to generate a standard curve for quantification. CgOrc1 and CgOrc2 were present in similar amounts (∼30 000 and ∼43 000 molecules/cell, respectively) throughout mitosis and G1 phase of the CHOC 400 cell cycle (Figure 5B) (CHOC 400 is a CHO cell line that contains ∼1000 tandemly integrated copies of the 273 kb DHFR gene amplicon). Measurements at other time points indicated that the levels of these proteins remained constant throughout the cell cycle (data not shown). To determine whether or not these proteins were present in the substrates that were used in the previous DNA replication assays (Figures 1 and 2), aliquots of nuclei prepared by digitonin lysis were assayed for CgOrc1 or CgOrc2 as described above (Figure 5C). The validity of using histones as an internal standard was confirmed by simultaneously immunoblotting α-actin. The higher percentage gel used in these assays revealed the presence of two CgOrc2 proteins, similar to what has been observed with Drosophila Orc2 (Huang et al., 1998). The results confirmed that the two hamster Orc proteins were present in similar amounts in nuclei prepared using digitonin, and in whole cells (Figure 5D). Taken together with the DNA replication assays, these data reveal that hamster Orc proteins are not functional during mitosis and early G1 phase. CgOrc1 and CgOrc2 differ in their association with chromatin as cells transit from mitosis to G1 phase To investigate whether the inability of Orc proteins to function during the M–G1 transition could result from an inability to bind stably to hamster chromatin, the amount of CgOrc1 and CgOrc2 that remained associated with nuclear pellets was determined by immunoblotting as described above except that cells were lysed with the non-ionic detergent Triton X-100, and then the nuclei were washed in a Triton X-100 buffered salts solution containing ATP. This procedure permeabilizes nuclei to large molecules and allows weakly bound proteins to be eluted from chromatin while stabilizing pre-RCs. Two different protocols, A and B, were tested (see Materials and methods) in case subtle differences affected the outcome. These protocols measure the amount of Orc bound to chromatin, because human Orc2 has been shown to be tightly bound to oligonucleosomes (Ritzi et al., 1998), and both CgOrc1 and CgOrc2 can be released from the pellet fraction by digestion with micrococcal nuclease (data not shown). CgOrc1 and CgOrc2 were quantified routinely in separate aliquots of the same lysate. The amount of CgOrc2 stably bound to chromatin during mitosis and G1 phase was constant at ∼2.6 ng/106 cells (∼24 000 molecules/cell) (Figure 6), or about half of the total CgOrc2 present in these cells. In contrast, the amount of CgOrc1 in the same extracts changed dramatically from an average ∼0.3 ng CgOrc1/106 cells during metaphase to an average of ∼4.1 ng CgOrc1/106 cells (∼26 000 molecules/cell) by 4 h into G1 phase (Figure 7). The amount of CgOrc1 stably bound to chromatin in metaphase cells varied among experiments from <0.1 to 1.3 ng/106 cells, while the amount of Orc1 in late G1 nuclei was comparatively stable. CgOrc1 that was not bound tightly to chromatin appeared to be unstable under these extraction conditions, since it was largely absent from the supernatant fractions, and was presumably degraded during sample preparation (data not shown). The same results were obtained using protocols A and B, and with cells that had been synchronized either with or without nocodazole present (Figure 7A). Figure 6.CgOrc2 remained tightly bound to chromatin during mitosis and G1 phase. Nuclei were prepared in the presence of Triton X-100, NaCl and ATP using protocol A or B (see Materials and methods). One aliquot was used to quantify the amount of CgOrc2 that remained bound, as described in Figure 5. Another aliquot was used to quantify the amount of CgOrc1 (see Figure 7). (A) The amount of CgOrc2 using protocol B was quantified as in Figure 5. (B) The CgOrc2:histone ratio in each gel lane was determined in four separate experiments (two by protocol A and two by protocol B). Each set of results was then normalized to a ratio of 1 for the 3 h time point, and then combined to give a mean ± SEM (filled squares). S phase was determined as in Figure 5D. Download figure Download PowerPoint Figure 7.CgOrc1 binds weakly to hamster chromatin during mitosis but strongly during early G1 phase. (A) CgOrc1 was quantified in aliquots of the same Triton X-100, NaCl, ATP prepared nuclei described in Figure 6. Cells were synchronized at metaphase in the presence (+ noc) or absence (− noc) of nocodazole. (B) The CgOrc1:histone ratio in each gel lane was determined, and then normalized to the maximum value in that experiment. The mean ± SEM for six experiments was determined (filled squares) and compared with the % CgORC activity (open squares), the reciprocal of the % XlOrc-dependence in Figure 4. Download figure Download PowerPoint These results revealed that individual hamster Orc proteins can behave differently during the transition from metaphase to G1 phase, because CgOrc2 binds tightly to chromatin throughout mitosis and G1 phase, whereas CgOrc1 binds weakly to chromatin during mitosis and early G1 phase, and then strongly during late G1 phase. This transition from weak to strong Orc1 binding coincided with the appearance of a functional hamster ORC in G1 nuclei (Figure 7B). Since measuring ORC activity required that the nuclei had to be incubated for 1 h in an XlOrc-depleted extract (Figure 1), ORC activity may appear more quickly than CgOrc1 binding to chromatin, because CgOrc1 may continue binding to chromatin in vitro. These results suggest that assembly of pre-RCs at specific chromosomal sites in mammalian nuclei begins with the stable binding of Orc1. Binding and activity of Mcm3 protein mimics Orc1 Previous studies in yeast and in Xenopus egg extracts have shown that Mcm proteins do not bind to chromatin before ORC binding (Romanowski et al., 1996 and references therein). To confirm that pre-RC assembly in hamster nuclei is delayed until G phase, Xenopus egg extract was depleted of XlMcm3 protein and then tested for its ability to initiate DNA replication in hamster G1 nuclei. Since XlMcm3 in Xenopus egg extracts exists as part of an Mcm2–7 complex, depletion of XlMcm3 depletes the other XlMcm proteins as well (Thommes et al., 1997). As previously observed with XlOrc-depleted extracts, XlMcm-depleted extracts were not able to initiate replication in Xenopus sperm chromatin, hamster metaphase chromatin or hamster early G1 nuclei, but were able to initiate replication in hamster late G1 nuclei (Figure 8B). The extent to which hamster nuclei were dependent on XlMcm proteins was inversely related to the amount of CgMcm3 protein stably bound to hamster chromatin (Figure 8). Chromatin-bound CgMcm3, which sometimes appeared as a doublet [probably due to differences in its phosphorylation (Todorov et al., 1995)], increased at least 9-fold as cells transited from mitosis to late G1 phase. Taken together, these results are consistent with the delayed binding of CgOrc1 to hamster chromatin. Figure 8.CgMcm3 bound weakly to hamster chromatin during mitosis but strongly during early G1 phase. (A) CgMcm3 was quantified in nuclei prepared with Triton X-100, NaCl and ATP using protocol A. The Mcm3:histone ratio in each gel lane was normalized to the maximum value (6 h after metaphase). (B) The relative amoun