|Home | About | Journals | Submit | Contact Us | Français|
A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2–7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2–7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.
Budding yeast origins of DNA replication are bound throughout the cell cycle by the Origin-Recognition Complex (ORC) (Bell and Stillman, 1992 ; Diffley and Cocker, 1992 ; Diffley et al., 1994 ; Santocanale and Diffley, 1996 ; Aparicio et al., 1997 ; Tanaka et al., 1997 ). During G1 phase, a larger prereplicative complex (pre-RC) is assembled around ORC, as evidenced by genomic footprinting (Diffley et al., 1994 ). It is likely that pre-RCs play a key role in the earliest stages of chromosome replication, since the initiation site of bidirectional replication at the chromosomal origin ARS1 has been found to lie in the center of the pre-RC footprint (Bielinsky and Gerbi, 1999 ). The formation of pre-RCs represents a key step in establishing the “replication competence” of an origin and is inhibited outside of G1-phase by cyclin-dependent kinase activity (Dahmann et al., 1995 ; Detweiler and Li, 1998 ), thereby ensuring that each origin is activated just once during S-phase, so that a single copy of the genome is made in each round of the cell cycle.
Until now, the protein components of the pre-RC, as defined by genomic footprinting, have remained poorly characterized. One candidate is the Cdc6 protein, which is essential for pre-RC formation at the end of mitosis and during G1-phase (Cocker et al., 1996 ; Santocanale and Diffley, 1996 ; Detweiler and Li, 1997 ). However, Cdc6 is degraded early in G1-phase (Piatti et al., 1995 ; Drury et al., 1997 ), whereas pre-RCs persist at origins until initiation occurs, or until the origin is replicated passively during S-phase, by replication forks from neighboring origins (Santocanale et al., 1999 ). Cdc6 is required for the six members of the MCM2–7 family to become associated with chromatin (Donovan et al., 1997 ; Liang and Stillman, 1997 ; Weinreich et al., 1999 ). MCM proteins are further candidates, therefore, for the proteins that make up the pre-RC; moreover, they play a key role in both the initiation and elongation stages of chromosome replication in budding yeast (Labib et al., 2000 ; Yan et al., 1993 ), and they have been shown to be components of “Replication Licensing Factor” in Xenopus egg extracts (Chong et al., 1995 ; Madine et al., 1995 ; Kubota et al., 1997 ; Thommes et al., 1997 ).
Experiments involving chromatin immunoprecipitation have shown that Cdc6 is essential for associating MCM proteins with origin-containing DNA (Aparicio et al., 1997 ; Tanaka et al., 1997 ). However, these experiments, which have a resolution of around 500 bp, do not distinguish between the possibility that MCM proteins are bound to origins as components of pre-RCs, or instead are associated with other sequences adjacent to the origin. Furthermore, an allele of CDC6 has been described, CDC6-d1, that supports partial pre-RC formation at ARS305 in the absence of bulk loading of MCM proteins onto chromatin (Perkins and Diffley, 1998 ). The role of MCM proteins in pre-RC formation, therefore, remains unclear.
Other proteins, such as Cdc45 and Sld3, are known to associate with early origins of DNA replication during G1 phase (Aparicio et al., 1999 ; Kamimura et al., 2001 ). It is unlikely that these proteins are core-components of pre-RCs, however, as they do not associate with late origins until S-phase, whereas pre-RC formation occurs at all origins at the end of mitosis. Furthermore, genomic footprinting of a cold-sensitive allele of CDC45, cdc45–1, has shown that cells accumulate at the restrictive temperature with the 2-μm origin of DNA replication in the prereplicative state (Owens et al., 1997 ).
In addition to their role in the initiation of chromosome replication, it has also been suggested that pre-RCs may be the source of a checkpoint signal, during G1 phase and S-phase, that inhibits premature entry into anaphase (Kelly et al., 1993 ; Maiorano et al., 1996 ; Piatti et al., 1995 ; van Brabant et al., 2001 ). If pre-RC formation is blocked, by depletion of Cdc6, anaphase occurs in the absence of S-phase (Piatti et al., 1995 ). Conversely, when the checkpoint is active–for example if cells are treated with the ribonucleotide-reductase inhibitor hydroxyurea, which reduces dNTP pools and inhibits the progression of DNA replication forks from early origins–pre-RCs are still present at late origins (Santocanale and Diffley, 1998 ). It has been proposed, therefore, that anaphase may not occur until pre-RCs are disassembled throughout the genome (van Brabant et al., 2001 ).
Alternatively, the checkpoint could be activated by events downstream of pre-RC formation, dependent upon initiation. This may involve proteins present at replication forks, aspects of the DNA structure of forks, or the presence of RNA primers in Okazaki fragments. Li and Deshaies (1993) proposed that these possibilities could be distinguished, by comparing the effects of inactivating a candidate protein, either before or after the establishment of DNA replication forks, and then by testing whether chromosome segregation occurs (Li and Deshaies, 1993 ). For example, the effects of inactivating pre-RCs in G1 phase or in hydroxyurea-arrested cells could be compared. Previous experiments with cdc6 mutants do not address this issue, as they allow us to examine only the effects of inhibiting pre-RC formation in G1-phase.
Here we use temperature-sensitive degron mutants and genomic footprinting to show, for the first time, that MCM proteins are essential components of the pre-RC in budding yeast. Degron mutants of MCM2–7 genes provide a tool that allows us to inactivate pre-RCs after their formation, either in G1-phase or during S-phase. We use these mutants to test the role of pre-RCs in the S-phase checkpoint.
The strains used in this study are based upon W303–1a and are listed in Table Table1.1. The construction of strains carrying either a degron allele of one of the MCM2–7 genes or a fusion of Mcm4 to green fluorescent protein (GFP) has been described previously, together with details of media composition and protocols for cell-cycle arrests (Labib et al., 1999 ; Labib et al., 2000 ).
To make pKL153, a PvuI fragment from pAFS91 (Straight et al., 1997 ), containing the TUB1- GFP gene fusion, was subcloned into PvuI digested pRS305 (Sikorski and Hieter, 1989 ). To direct integration of this plasmid to the LEU2 locus, the plasmid was linearized with the restriction enzyme AflII before transformation.
Genomic footprinting at the chromosomal origin ARS305 was performed as described previously (Noton and Diffley, 2000 ; Perkins and Diffley, 1998 ). Protocols for microscopy and flow cytometry were as described (Labib et al., 1999 ). The rabbit polyclonal antibody JD145, kindly provided by Corrado Santocanale, was used at a dilution of 1/1000 to detect Rad53 protein.
In budding yeast MCM2–7 proteins accumulate in the nucleus at the end of mitosis, when pre-RCs form, and are excluded from the nucleus as they are displaced from chromatin during S-phase (Dalton and Whitbread, 1995 ; Hennessy et al., 1990 ; Labib et al., 1999 ; Nguyen et al., 2000 ; Yan et al., 1993 ). Addition of an exogenous nuclear localization signal to any of the MCM2–7 proteins prevents nuclear exclusion of all the others (Nguyen et al., 2000 ), indicating that they associate with each other between S-phase and the end of mitosis. By examining the localization of a fusion of Mcm4 to GFP (Mcm4-GFP), we have taken a converse approach to address whether MCM2–7 proteins also associate with each other during late mitosis and G1 phase, as cells pass through mitosis and into G1 phase in the absence of another member of the MCM2–7 family (Figure (Figure1A).1A). To do this, we used strains in which the only copy of a particular MCM gene was fused to the temperature-sensitive degron cassette (Labib et al., 2000 ). Proteolysis of degron-fusion proteins involves recognition by the Ubr1 protein, followed by polyubiquitylation of lysine residues in the degron cassette, and is stimulated at high temperatures (Dohmen et al., 1994 ). To improve the efficiency of degradation, and to provide a further level of regulation, we used strains in which the only copy of the UBR1 gene is expressed from the GAL1,10 promoter (Labib et al., 2000 ).
Degron mutants of MCM2, 3, 6, or 7, together with a control strain, were grown at 24°C in the absence of UBR1 expression. Cells were arrested in G2/M with the microtubule-depolymerising drug nocodazole, and the cultures were split in two. Expression of UBR1 was induced for 45 min in one half, and each culture was then shifted to 37°C for an additional 45 min. Proteolysis of the degron-fusion proteins occurred specifically in the cultures expressing Ubr1, without affecting the stability of the other MCM2–7 proteins (Figure (Figure1)1) (Labib et al., 2000 ). Cells were then released into fresh medium containing α-factor mating pheromone instead of nocodazole, so that they completed mitosis and arrested in the subsequent G1-phase (which was confirmed microscopically).
Mcm4-GFP was predominantly cytoplasmic in G2/M arrested cells and then accumulated in the nucleus as the control strain completed mitosis and entered G1-phase (Figure (Figure1B,1B, control), either in the presence or absence of Ubr1 protein (YPGal and YPRaff, respectively). Mcm4-GFP also accumulated in the nucleus of the mcm2, 3, 6, 7 degron mutants, when cells passed through mitosis at 37°C in the absence of Ubr1 protein (Figure (Figure1B,1B, YPRaff). In contrast, Mcm4-GFP did not accumulate in the nucleus of cells lacking either Mcm2, 3, 6, or 7 proteins (Figure (Figure1B,1B, YPGal). This indicates that MCM2–7 proteins interact with each other during the transition between late mitosis and early G1-phase.
We also examined the effects of degrading Mcm2, 3, 6, or 7 in G1-arrested cells, after nuclear accumulation of MCM proteins and pre-RC formation had already occurred (Figure (Figure2A).2A). Cells were arrested in G1 phase at 24°C, and once again the cultures were split in two, before induction of UBR1 expression in one half. Localization of Mcm4-GFP was examined both before and after shifting the cultures to 37°C for 1 hour. In all strains, Mcm4-GFP was nuclear at 24°C in G1-arrested cells (Figure (Figure2B,2B, stages 1 and 3). On shifting cells to 37°C in the absence of Ubr1 protein, Mcm4-GFP remained nuclear in all strains (Figure (Figure2B,2B, stage 4). However, degradation of either Mcm2, 3, 6, or 7 by shifting cells to 37°C in the presence of Ubr1 protein caused loss of Mcm4-GFP from the nucleus (Figure (Figure2B,2B, stage 2) without affecting the level of Mcm4-GFP protein (Figure (Figure2C).2C).
Taken together with the results of Nguyen et al., the preceding experiments indicate that MCM proteins interact with each other throughout the budding yeast cell cycle. Moreover, our data show that this interaction is essential for nuclear accumulation to occur during late mitosis and G1-phase, when MCM2–7 proteins are loaded onto chromatin at origins of DNA replication.
We used mcm degron mutants to address directly the role of MCM proteins in pre-RC formation at ARS305. From the time of initiation, during early S-phase, until late mitosis, the origin is in the postreplicative state, characterized by three ORC-induced DNase I hypersensitive sites (sites 1–3, Figure Figure3,3, Control, stage 1). During G1 phase, the larger prereplicative complex at this origin is characterized by suppression of the three ORC-induced hypersensitive sites in the genomic footprint, together with an extended region of protection from DNase I digestion adjacent to the ORC binding site and induction of a G1-specific hypersensitive site (Figure (Figure3,3, Control, stage 2, (Noton and Diffley, 2000 ; Perkins and Diffley, 1998 )).
To test whether MCM2–7 proteins are required for pre-RC formation at ARS305, we grew mcm4-td, mcm7-td, and a control strain in the absence of UBR1 expression at 24°C, and we arrested cells in G2/M with the microtubule depolymerising drug nocodazole. Expression of UBR1 was then induced for 30 min, and cells were shifted to 37°C for 45 min to degrade the degron-fusion proteins Mcm4-td and Mcm7-td. At this stage, ARS305 was in the postreplicative state in all three strains (Figure (Figure3,3, stage 1). Cells were then released into fresh medium at 37°C for 2 hours, in the presence of α-factor mating pheromone, so that they completed mitosis before arresting in the subsequent G1 phase. In the control strain, pre-RC formation could be observed at ARS305 (Figure (Figure3,3, Control, stage 2). In the absence of Mcm4 or Mcm7, however, pre-RC formation did not occur, and instead the origin remained in the postreplicative state (Figure (Figure3,3, mcm4-td and mcm7-td, stage 2). This shows that MCM function is essential for pre-RC formation to occur as cells pass through mitosis and into G1 phase.
To provide stronger evidence that MCM2–7 proteins are components of the pre-RC at ARS305, rather than simply being required for its formation, we tested the effects of degrading Mcm4 or Mcm7 in G1 cells, after pre-RC formation had already occurred. The same three strains as above were grown at 24°C in the absence of UBR1 expression, and cells were arrested in G1 phase with α-factor. At this stage of the experiment, pre-RC formation at ARS305 could be observed in all three strains (Figure (Figure4,4, stage 1). Expression of UBR1 was then induced for 30 min, and the cultures were split in two. One half was shifted to 37°C for 1 hour to induce degradation of Mcm4-td and Mcm7-td proteins (Figure (Figure4,4, stage 2). As a control, the other half of each culture was left at 24°C for the same period of time (Figure (Figure4,4, stage 3). G1-arrest was maintained throughout the experiment. Degradation of either Mcm4 or Mcm7 caused the origin to revert from the prereplicative to the postreplicative state (Figure (Figure4,4, compare stages 2 and 3 for each strain). All three features of the pre-RC at ARS305 were lost upon degradation of an MCM protein: the G1-specific hypersensitive site disappeared, the three ORC-induced hypersensitive sites reappeared, and the region of protection from DNase I digestion, adjacent to the ORC-binding site, was also lost (Figure (Figure4).4).
These experiments show that MCMs are essential for formation and maintenance of the pre-RC at ARS305, suggesting that the pre-RC footprint may represent, in fact, the MCM-binding site at budding yeast origins of replication, adjacent to ORC.
The preceding experiments show that mcm degron mutants provide a tool with which we can degrade preexisting pre-RCs at origins of DNA replication. This allows us to test the role of pre-RCs in the S-phase checkpoint, after the approach suggested by Li and Deschaies (1993) . First, we examined the effects of degrading an MCM protein before the establishment of DNA replication forks. Cultures of mcm4-td, mcm7-td, and a control strain were grown at 24°C in the absence of UBR1 expression, and G1-arrested cells, lacking pre-RCs (mcm4-td and mcm7-td) or containing pre-RCs (control), were generated at 37°C, exactly as described above for the experiment in Figure Figure3.3. Cells were then released from G1 arrest at 37°C into fresh medium, and samples were taken every 20 min to follow DNA content and progression through mitosis. One half of the control culture was released from G1 arrest into medium containing 0.2 M hydroxyurea (HU), as a positive control for activation of the checkpoint that inhibits mitosis in response to incomplete S-phase.
The control strain completed S-phase and mitosis rapidly in the absence of HU, before entering the next cell cycle (Figure (Figure5,5, control). In the presence of HU, S-phase was blocked, and activation of the checkpoint prevented anaphase (Figure (Figure5,5, control +HU). Degradation of Mcm7-td protein prevented S-phase, but it did not block the subsequent anaphase, showing that checkpoint activation was defective in the absence of pre-RCs (Figure (Figure5,5, mcm7-td). A smaller proportion of cells with divided nuclei was seen after degradation of Mcm4-td protein (Figure (Figure5B),5B), probably reflecting the slightly leakier nature of the mcm4-td allele (compare the flow cytometry profiles of mcm4-td and mcm7-td in Figure Figure5A).5A). These data are consistent with a previous report of an allele of MCM3, mcm3–10, for which a proportion of cells undergo nuclear division without completing chromosome replication (Toyn et al., 1995 ).
These experiments show that anaphase can occur in the absence of S-phase, due to a failure in checkpoint activation, if prereplicative MCM2–7 complexes are degraded before establishment of DNA replication forks. We then examined the effects of degrading an MCM protein after the establishment of forks from early origins of DNA replication. The same three strains as before were arrested in G1 phase in the absence of UBR1 expression, before releasing into fresh medium containing 0.2 M HU. We have shown previously that early origins of replication are activated efficiently in mcm degron mutants under such conditions (Labib et al., 2000 ). Expression of UBR1 was then induced for 45 min and the cultures shifted to 37°C in the continued presence of HU to induce degradation of Mcm4-td and Mcm7-td proteins. Cells were then released into fresh medium at 37°C that lacked HU, and progression through S-phase and mitosis was monitored every 20 min.
The control strain completed S-phase rapidly upon release from HU and then proceeded through a synchronous round of nuclear division (Figure (Figure6,6, control). Degradation of Mcm4-td or Mcm7-td prevented continued DNA synthesis (Figure (Figure6A),6A), as we have reported previously (Labib et al., 2000 ), due to a defect in DNA replication fork progression during the elongation phase of chromosome replication. However, nuclear division did not occur in cells lacking Mcm4 or Mcm7 proteins, either before or after release from HU, indicating that the checkpoint remained intact (Figure (Figure6B,6B, C). This was confirmed by examination of the phosphorylation status of the Rad53 protein kinase, an important transducer of the checkpoint signal, which remained in its active, hyperphosphorylated form upon release from HU in the absence of Mcm4 or Mcm7 (Figure (Figure6D).6D). This indicates that loss of MCM proteins, after the establishment of replication forks, actually promotes checkpoint inhibition of anaphase, by inhibiting the progression of replication forks during the elongation phase of chromosome replication. Just as HU blocks the progression of replication forks, and so anaphase, by a Rad9-independent mechanism (Weinert et al., 1994 ), so too the inhibition of nuclear division, seen when elongation is blocked by MCM depletion, is also independent of Rad9 function (Figure (Figure7).7).
Taken together, the preceding experiments indicate that MCM2–7 proteins do not play a direct role in checkpoint control during S-phase, either in pre-RCs or during elongation. Instead, pre-RCs play an indirect role, insofar as they are essential for initiation and the establishment of replication forks. Inactivation of MCM proteins during S-phase prevents entry into anaphase by inhibiting the progression of DNA replication forks.
The pre-RC is assembled over and around the ORC-binding site at origins of DNA replication in budding yeast. PreRC formation and maintenance require both Cdc6 (Cocker et al., 1996 ; Santocanale and Diffley, 1996 ) and the MCM2–7 complex (this study). As Cdc6 is required for the association of MCM2–7 proteins with chromatin, and as pre-RC formation cannot occur in cells containing Cdc6 but not MCM proteins, our data suggest that the prereplicative footprint represents the binding site of MCM2–7 proteins alongside ORC.
MCM2–7 proteins have been estimated to be between 20 and 100 times more abundant than ORC, Cdc6, or the number of origins of DNA replication (Lei et al., 1996 ; Donovan et al., 1997 ), and a significant proportion is associated with chromatin during G1-phase. The reason for the high relative-abundance of MCM proteins remains unclear. It is interesting to note that a mutant allele of CDC6, CDC6-d1, supports the formation of a partial prereplicative footprint at ARS305 but does not support the loading of wild-type levels of MCM2–7 proteins onto chromatin (Perkins and Diffley, 1998 ). The partial pre-RC induced by Cdc6-d1 protein produces suppression of ORC-induced hypersensitive sites 1 and 2 (see Figures Figures11 and and2)2) but does not cause suppression of the third ORC-induced hypersensitive site or protection of the adjacent region from DNase I digestion (Perkins and Diffley, 1998 ). Because all aspects of the pre-RC footprint at ARS305 are MCM-dependent, it is possible that the partial pre-RC and the full pre-RC differ quantitatively in the number of MCM2–7 complexes bound to the origin. For example, the partial pre-RC may contain a single MCM2–7 complex, and generation of the full pre-RC at ARS305 may require the binding of multiple MCM2–7 complexes. It is worth noting that, at a very late and normally silent origin such as ARS301, the pre-RC footprint involves suppression of two ORC-induced hypersensitive sites without significant protection of adjacent regions (Santocanale and Diffley, 1996 ). Perhaps one important difference between early origins, such as ARS305 and very late origins, such as ARS301, is that the former are bound by more MCM2–7 complexes than the latter. It is likely, however, that other factors also contribute to the determination of origin-timing, as proximity to a telomere delays activation of a normally-early origin without changing the prereplicative footprint (Santocanale and Diffley, 1998 ).
Our data, together with those of Nguyen et al. (Nguyen et al., 2000 ), indicate that budding yeast MCM2–7 proteins interact with each other in vivo, during late mitosis, G1-phase, and after S-phase. Furthermore, we show that this interaction is essential for nuclear accumulation of MCM proteins during the period of the cell cycle when they are assembled into pre-RCs, as previously reported for fission yeast (Pasion and Forsburg, 1999 ). We have also shown previously that Mcm2, 3, 4, 6, and 7 proteins are required during S-phase for the elongation phase of chromosome replication (Labib et al., 2000 ). Taken together, these experiments reinforce the notion that the active form of MCM2–7 proteins, throughout the cell cycle, is likely to be a heterohexamer.
Our experiments show that, once replication forks have been established from early origins, MCM2–7 proteins, and therefore pre-RCs, are not required to inhibit anaphase in response to incomplete chromosome replication. Inhibition of the progression of DNA replication forks, either by HU treatment or by MCM-depletion after initiation, blocks entry into anaphase. In both cases, hyperphosphorylation of Rad53 is maintained, and anaphase is inhibited in a Rad9-independent manner. It appears that stalling of replication forks, rather than presence of MCM2–7 proteins, or pre-RCs, is important for the checkpoint inhibiting mitosis in response to incomplete replication.
Several studies, however, have reported that other replication proteins, such as RF-C (Sugimoto et al., 1997 ; Noskov et al., 1998 ; Reynolds et al., 1999 ; Shimada et al., 1999 ) or the budding yeast Dpb11 protein and its fission yeast homologue Cut5 (Saka and Yanagida, 1993 ; Saka et al., 1994 ; Araki et al., 1995 ; McFarlane et al., 1997 ; Wang and Elledge, 1999 ) are required to maintain checkpoint inhibition of mitosis in HU-arrested cells, suggesting that these proteins may indeed play a role in checkpoint control. But it remains to be shown that activation of early origins and replication fork establishment have occurred normally in these experiments. Failure to establish replication forks, due to the combination of HU and the defective nature of a particular conditional allele chosen for such an experiment could cause entry into anaphase without the test protein having a direct role in checkpoint control.
It is worth noting that mitosis occurs with very similar timing, both in wild-type cells, and also in cells that segregate their chromosomes in the absence of DNA replication (this study, Piatti et al., 1995 ; Tercero et al., 2000 ). It is likely, therefore, that the timing of anaphase in budding yeast is determined by a second mechanism, distinct from the checkpoint that blocks mitosis in response to incomplete chromosome replication.
We favor the view that some aspect of the structure of replication forks may be sensed by checkpoint proteins, leading to the generation of the checkpoint signal. It has been argued that this may involve detection of the RNA primer present at the beginning of Okazaki fragments (Michael et al., 2000 ), but this view is not consistent with experiments implicating RF-C in checkpoint control, as RF-C acts after primer formation, and it is not clear how mutation of RF-C would affect the presence or absence of RNA primers in Okazaki fragments.
Our experiments suggest one approach to addressing these issues in the future, by making degron mutants of other replication proteins such as primase and RF-C and by comparing the effects of degrading these proteins in HU-arrested cells after confirming that establishment of replication forks from early origins has indeed taken place.
We thank Andrew Murray's laboratory for providing us with the plasmid pAFS91, Maria Ángeles de la Torre-Ruiz (ICRF Clare Hall Laboratories) for the rad9Δ strain, and Corrado Santocanale (ICRF Clare Hall Laboratories) for the antibody JD145. This work was funded by the Imperial Cancer Research Fund and by the Cancer Research Campaign.