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REVIEW

Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

	Abstract

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
In eukaryotic cells, prereplication complexes (pre-RCs) are assembled on chromatin in the G1 phase, rendering origins of DNA replication competent to initiate DNA synthesis. When DNA replication commences in S phase, pre-RCs are disassembled, and multiple initiations from the same origin do not occur because new rounds of pre-RC assembly are inhibited. In most experimental organisms, multiple mechanisms that prevent pre-RC assembly have now been identified, and rereplication within the same cell cycle can be induced through defined perturbations of these mechanisms. This review summarizes the diverse array of inhibitory pathways used by different organisms to prevent pre-RC assembly, and focuses on the challenge of understanding how in any one cell type, various mechanisms cooperate to strictly enforce once per cell cycle regulation of DNA replication.

[Keywords: Cdt1; Cul4; DNA replication; MCM2–7; ORC; SCF]

	The two-state model for cell cycle regulation of DNA replication

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
Early insights into the regulation of eukaryotic DNA replication came from cell fusion experiments (Rao and Johnson 1970), which showed that union of an S-phase cell with a G1 cell accelerates the rate at which the latter enters S phase. In contrast, G2 cells are refractory to this stimulation. These results suggested that the initiation of DNA synthesis requires a positive, diffusible S-phase-promoting activity, and that G1 but not G2-phase cells are competent to respond to this signal. Building on these observations and on their own experiments in Xenopus egg extracts, Blow and Laskey (1988) developed the "licensing" model, in which a licensing factor that is required for replication initiation binds to chromatin in G1. At the G1/S transition, the factor is inactivated, and it cannot be replenished until cells pass through mitosis. These ideas were extended by in vivo footprinting experiments in yeast that showed that origins of replication alternate between two distinct states (Diffley et al. 1994). In G1, all origins exhibit a "prereplicative" pattern. In contrast, from the moment of initiation until passage through mitosis, the origin resides in a "post-replicative" state. Concurrently, experiments by Nurse and colleagues (Broek et al. 1991; Hayles et al. 1994) showed that cyclin-dependent kinases (CDKs) play a dual role in regulating DNA replication, being required not only to trigger replication initiation but also to limit DNA replication to a single round per cell cycle. When CDK activity is inhibited in G2 phase, origins of replication revert to the prereplicative state and reinitiation occurs (Dahmann et al. 1995; Piatti et al. 1996). Finally, the identification of the origin recognition complex (ORC), Cdc6, Cdt1, and the minichromosome maintenance (MCM) complex as licensing activities and/or key components of prereplication complexes (pre-RCs) gave molecular definition to the replication factors whose activity is controlled during the cell cycle (for review, see Bell and Dutta 2002).

These seminal experiments coalesced into an elegant model for the cell cycle regulation of DNA replication, the essence of which is that DNA replication occurs in two discrete steps, which are closely correlated with oscillations in CDK activity (see Fig. 1A; Diffley 1996). The first step occurs soon after M-CDK activity drops upon exit from mitosis, and it involves the assembly of a pre-RC at each origin via the ordered binding of at least four factors, ORC, Cdc6, Cdt1, and MCM2–7. The process of pre-RC assembly is often referred to as "licensing." The second step, replication initiation, is triggered by the increase in S-phase CDK activity (S-CDK), which occurs at the G1/S transition. When replication initiates, the pre-RC reverts to a post-replicative state due to loss of the MCM2–7 complex. Multiple initiations from the same origin do not occur because once cells pass through the G1/S transition, they cannot load new MCM2–7 complexes onto origins. In short, the cell cycle oscillates between two functional states. During the first state, which covers G1 phase and is characterized by low CDK activity, pre-RCs are assembled but cannot undergo initiation. In the second state, which spans S, G2, and M phases, high CDK activity allows replication to initiate, but it also prevents de novo pre-RC assembly. This separation of initiation into two distinct phases, only the first of which is blocked by CDK activity, and the disassembly of pre-RCs upon initiation, together ensure that no origin of DNA replication can initiate more than once per cell cycle. An important corollary to this model is that the inhibitory mechanisms, which prevent de novo MCM2–7 recruitment have no effect on the stability of existing pre-RCs. Thus, at origins that initiate DNA replication late in S phase, pre-RCs persist on chromatin for extended periods in an environment that is refractory to new pre-RC formation.

View larger version (42K): [in this window] [in a new window]   Figure 1. Two-step model for the cell cycle regulation of eukaryotic DNA replication. (A) The events that occur at origins of DNA replication at different stages of the cell cycle are shown. The green bars indicate when in the cell cycle licensing and initiation, respectively, are allowed. (B) Oscillations in APC and CDK activity during the cell cycle are indicated.

	Step one: licensing origins of replication via pre-RC formation

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
All known mechanisms that restrict DNA replication to a single round per cell cycle inhibit the first phase of replication initiation, origin licensing (for review, see Bell and Dutta 2002; Blow and Dutta 2005). Licensing begins with DNA binding of the ORC, a six-subunit AAA⁺ ATPase (Fig. 2). In budding and fission yeasts, ORC binds chromatin throughout the cell cycle, but in higher eukaryotes ORC phosphorylation during mitosis causes its transient release from chromatin (for review, see DePamphilis 2005). In Saccharomyces cerevisiae, ATP binding by ORC is required for the specific recognition of origin DNA sequences, and in multicellular eukaryotes, where ORC exhibits little or no sequence specificity, ATP binding is required for efficient interaction with any DNA sequence (for review, see Cvetic and Walter 2005). Chromatin-bound ORC recruits two additional proteins, Cdc6, another AAA⁺ ATPase, and Cdt1, a coiled-coil domain protein. Together, ORC, Cdc6, and Cdt1 facilitate chromatin loading of the MCM2–7 complex, which almost certainly functions as the replicative DNA helicase (see below). Recent experiments in licensing-competent yeast extracts suggest distinct functions for the ATPase activities of ORC and Cdc6 in MCM2–7 recruitment (Bowers et al. 2004; Randell et al. 2006). Thus, when ATP hydrolysis by Cdc6 is blocked, MCM2–7 binds origin DNA but remains loosely associated. In contrast, in the absence of ATP hydrolysis by ORC, MCM2–7 is loaded and associates tightly with the origin, but the number of MCM2–7 complexes that bind to each origin is reduced. Interestingly, S. cerevisiae Cdt1 forms a complex with MCM2–7 in solution, but Cdt1 is only stably bound to the origin when ATP hydrolysis by Cdc6 is blocked (Tanaka and Diffley 2002; Randell et al. 2006). Together, these observations support a model in which Cdc6 associates with ORC, after which MCM2–7 is escorted to the origin via Cdt1 (Fig. 2). ATP hydrolysis by Cdc6 causes a conformational change in MCM2–7, which induces tight MCM2–7 binding to DNA and displacement of Cdt1. Finally, ATP hydrolysis by ORC inaugurates a new round of MCM2–7 loading. This cycle appears to be repeated multiple times, yielding pre-RCs containing many MCM2–7 complexes (see below). Unlike yeast Cdt1, metazoan Cdt1 does not form a stable complex with MCM2–7, but direct binding of Cdt1 to MCM2–7 is nevertheless thought to be essential for pre-RC assembly (Yanagi et al. 2002; Cook et al. 2004; Ferenbach et al. 2005).

View larger version (22K): [in this window] [in a new window]   Figure 2. Model for the mechanism of pre-RC assembly in yeast. ORC:ATP binds to origin DNA (1) and recruits Cdc6:ATP (2). (3) ORC and Cdc6 then recruit a complex of MCM2–7 and Cdt1. (4) Upon ATP hydrolysis by Cdc6, MCM2–7 binds tightly to DNA, possibly by encircling the duplex. (5) ORC hyrdrolyzes ATP. The resulting conformational change releases MCM2–7 and presumably leads to release of the ORC complex from DNA. The cycle begins anew when a new ORC:ATP complex binds to the origin. Repetition of these five steps leads to the recruitment of multiple MCM2–7 complexes to a single origin of DNA replication.

Interestingly, recent evidence suggests that MCM2–7 may not unwind DNA on its own. MCM2–7 has been purified in a ternary complex with two other replication initiation factors, Cdc45 and GINS (Kubota et al. 2003; Masuda et al. 2003; Gambus et al. 2006; Moyer et al. 2006), and highly purified preparations of this complex exhibit helicase activity (Moyer et al. 2006). Importantly, Cdc45 and GINS are associated with replication forks and are required for their progression (Aparicio et al. 1997; Tercero et al. 2000; Takayama et al. 2003; Pacek and Walter 2004; Pacek et al. 2006). Moreover, inhibition of Cdc45 blocks DNA unwinding at the replication fork in Xenopus egg extracts, and both Cdc45 and GINS associate with the uncoupled DNA helicase (Pacek and Walter 2004; Pacek et al. 2006). Because Cdc45 and GINS lack ATPase motifs, the data collectively suggest that MCM2–7 is the engine that stimulates DNA unwinding, while Cdc45 and GINS play auxiliary functions.

Considering that MCM2–7 likely functions as the replicative DNA helicase, it is puzzling that in most organisms, between five and 40 MCM2–7 complexes bind to each origin of DNA replication (Fig. 2; Randell et al. 2006; for review see Takahashi et al. 2005). In Xenopus egg extracts, these multiple MCM2–7 complexes are widely distributed on DNA, and they all appear to be functional (Edwards et al. 2002; Harvey and Newport 2003; Woodward et al. 2006). However, only a small subset of the chromatin-bound complexes is normally required to support efficient DNA replication in this system (Mahbubani et al. 1997; Edwards et al. 2002; Woodward et al. 2006). In yeast, mutations in ORC that limit the number of MCM2–7 complexes loaded onto each origin of replication are lethal (Randell et al. 2006), but the reason underlying lethality is unknown. Presently, it is unclear why so many MCM2–7 complexes are loaded onto origins, although several models have been proposed that suggest that the "latent" MCM2–7 complexes are activated during replicative crises (Edwards et al. 2002; Hyrien et al. 2003; Woodward et al. 2006). Whatever their function, all the MCM2–7 complexes associated with chromatin must be removed during the first round of DNA replication to prevent reinitiation. Most likely, passage of the DNA replication fork displaces latent MCM2–7 complexes (Brewer and Fangman 1993; Santocanale et al. 1999).

The dynamics of pre-RCs are highly relevant for the cell cycle regulation of DNA replication. MCM2–7 complexes bind very stably to DNA, being resistant to extraction by high salt (Donovan et al. 1997; Rowles et al. 1999; Edwards et al. 2002; Bowers et al. 2004), consistent with the observation that MCM2–7 complexes persist at origins for extended periods of the cell cycle, especially at late origins. The molecular basis for MCM2–7’s tight grip on DNA is not understood, although it has been proposed that MCM2–7 may encircle double-stranded DNA within pre-RCs (Mendez and Stillman 2003; Takahashi et al. 2005). This idea is based in part on the apparent similarity between pre-RC assembly and the deposition of polymerase processivity factors onto DNA by clamp loaders (Perkins and Diffley 1998; Randell et al. 2006). Importantly, after MCM2–7 is loaded, ORC, Cdc6, and Cdt1 are no longer required for initiation of DNA replication, indicating that their primary function in DNA replication is to deliver MCM2–7 to origins (Muzi Falconi et al. 1996; Hua and Newport 1998; Duncker et al. 1999; Rowles et al. 1999; Maiorano et al. 2000; Shimada et al. 2002). This feature of pre-RCs is crucial, because it means that ORC, Cdc6, and Cdt1 can be inactivated at the G1/S transition to prevent de novo pre-RC assembly without affecting subsequent initiation. Interestingly, it appears that binding of the MCM2–7 complex to origins of replication stimulates the dissociation of ORC, Cdc6, and Cdt1 (Rowles and Blow 1997; Harvey and Newport 2003; Randell et al. 2006). Dissociation from pre-RCs should liberate these factors for assembly of new pre-RCs elsewhere, while perhaps also rendering them more accessible for inactivation by proteolysis and other mechanisms when cells enter S phase (see below).

	Step two: initiation of DNA replication and origin inactivation

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
Origins that are licensed during the G1 phase do not initiate DNA synthesis until they are acted on by a plethora of other initiation factors in S phase (for recent reviews, see Bell and Dutta 2002; Takeda and Dutta 2005; Walter and Araki 2006). The key event in the initiation of DNA replication is thought to be activation of MCM2–7 helicase activity, which requires assembly of the Cdc45–GINS–MCM2–7 complex. Briefly, two protein kinases, CDK and DDK (Dbf4 and Drf1-dependent kinase) cooperate with many other factors, including Mcm10 and Dpb11, to deposit Cdc45 and GINS onto the MCM2–7 complex (Sld2 and Sld3, two proteins implicated in this process in yeast, remain to be clearly identified in metazoans). Recent evidence supports the long-standing idea that DDK functions by directly phosphorylating the MCM2–7 complex, an event believed to facilitate its interaction with Cdc45 (Masai et al. 2006; Sheu and Stillman 2006; Tsuji et al. 2006). While two CDK substrates, Sld2 and Sld3, have been identified in yeast (Masumoto et al. 2002; Tanaka et al. 2007; Zegerman and Diffley 2007), the targets of this protein kinase are still unknown in metazoans. Once the origin is unwound, DNA polymerase /primase is recruited, synthesizing an 10 nucleotide RNA primer, which it further extends with 30 deoxynucleotides. The RNA–DNA primer is recognized by replication factor C (RFC), which displaces DNA polymerase /primase and in turn recruits PCNA, a ring-shaped trimer that encircles DNA and functions as the processivity factor for DNA polymerase (Maga and Hubscher 2003).

The two-step, cell cycle regulation of DNA replication can now be described from the perspective of the replicative DNA helicase. In the G1 phase, origins of DNA replication are rendered competent when the core of the helicase, the MCM2–7 complex, is assembled into pre-RCs. In S phase, helicase assembly is completed with the recruitment of Cdc45 and GINS. During initiation, the pre-RC is dismantled when the helicase moves away from the origin. Critically, new MCM2–7 complexes cannot be recruited once cells enter S phase, so helicase reassembly, which would be required for reinitiation, is not possible. In contrast, the subsequent steps, Cdc45 and GINS recruitment, are promoted in S phase. Indeed, transient destruction of MCM2–7 in mid-S phase causes permanent cell cycle arrest, whereas after transient elimination of Cdc45, DNA replication resumes (Labib et al. 2000; Tercero et al. 2000). By targeting the replicative DNA helicase, cells block rereplication at the earliest enzymatic step of chromosome duplication, thereby minimizing the possibility of genomic instability.

	Anaphase-promoting complex (APC): the master regulator of licensing

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
The cell cycle regulation of DNA replication is inextricably connected to the cell cycle engine, whose major feature is the periodic oscillation of CDK activity (Fig. 1B; for review, see Morgan 2007). CDKs consist of a catalytic subunit that is activated upon binding to a cyclin subunit, which also confers substrate specificity. Cyclins that control cell proliferation can be grouped into four categories: the G1 cyclins, the G1/S cyclins, the S-phase cyclins, and the M-phase cyclins. With few exceptions, only the S- and M-phase CDKs inhibit pre-RC formation, so it is important to consider how their abundance is regulated. The exit from mitosis, which is marked by destruction of M-CDK activity, is stimulated by a multisubunit E3 ubiquitin ligase called the APC. M-CDKs promote their own destruction by phosphorylating a version of the APC that contains the activator protein Cdc20 (APC^Cdc20). APC^Cdc20 then targets Cyclins A and B for destruction. Importantly, most eukaryotic cells also express, APC^Cdh1, whose activator subunit Cdh1 is inhibited by M-CDK activity. Thus, when M-phase cyclins are destroyed, and APC^Cdc20 becomes inactive, APC^Cdh1 steps in. Like APC^Cdc20, APC^Cdh1 destroys M- and S-phase-specific cyclins, and stabilizes CDK inhibitors (CKIs). APC^Cdh1 thereby perpetuates a G1 state that is characterized by low S- and M-CDK activity. The G1 phase ends when growth signals activate expression of G1/S cyclins. G1/S-CDKs, which are not substrates of APC^Cdh1, phosphorylate and switch off APC^Cdh1. APC^Cdh1 activity is also blocked in S and G2 phase by Emi1. Subsequently, S- and M-phase cyclins can reaccumulate and CKIs are destroyed. In yeast, activation of APC at the end of mitosis sets the stage for origin licensing by establishing a window of low S/M-CDK activity. In higher eukaryotes, the APC additionally targets the licensing inhibitor Geminin (see below). In summary, the APC is a master regulator whose activity determines whether cells are in a state that is permissive or restrictive for pre-RC formation.

	Rereplication, failed mitosis, endoreduplication, and gene amplification

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
The ploidy of eukaryotic cells can change for a variety of reasons. First, during rereplication, replication origins initiate DNA synthesis more than once but there is no coordination between reinitiation events, and thus, the resulting increase in ploidy is usually partial. Second, if mitosis fails due to a defect in cytokinesis, cyclins are still destroyed by the APC, allowing pre-RC reassembly and progression into a new cell cycle. Mitotic failure causes a doubling of DNA content. Rereplication and mitotic failure are generally not programmed events, but rather result spontaneously from defects in the cell cycle machinery. Third, cells can undergo endoreduplication, which usually involves consecutive and complete S phases that are not separated by mitosis. Endocycles occur in many metazoans during normal development. Fourth, during gene amplification, specific segments of the chromosome undergo repeated initiation events, leading to increased copy number of particular loci. We collectively refer to these four phenomena as "overreplication." This review focuses primarily on the mechanisms that prevent rereplication.

	Redundancy and the regulation of origin firing

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
It is generally assumed (but not proven) that reinitiation from even a single origin of DNA replication is undesirable and possibly fatal. Considering that some eukaryotic cells initiate DNA replication at up to 300,000 sites during a single S phase, it follows that, in some cases, cells must reduce the incidence of origin refiring to <0.0003%. This requirement for virtually absolute repression of rereplication is probably why cells have developed multiple mechanisms to inhibit pre-RC assembly. The presence of multiple inhibitory mechanisms raises conceptual questions concerning redundancy that are intertwined with the details of experimental methods. Thus, if at least two separate mechanisms must be disrupted to detect rereplication in a particular system, and the limit of detection is 1% of the genome rereplicated, then it follows that each individual mechanism is by itself >99% efficient. When acting together, these two mechanisms would achieve at least 99.99% repression. In this scenario, the mechanisms are overlapping, and they should not be considered functionally redundant unless each alone is sufficient to maintain genome stability over many generations. In some instances, inhibitory mechanisms have been characterized that are not essential for viability. Such mechanisms are considered dispensable. As discussed below, cells probably achieve the desired level of origin repression by employing multiple overlapping inhibitory mechanisms whose combined, multiplicative effect is adequate to support viability.

	Strategies to prevent rereplication in different eukaryotes

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
Using defined manipulations, rereplication can now be induced in most major systems used to study eukaryotic DNA replication. As described in the sections below, these experiments indicate that all eukaryotes employ multiple mechanisms to prevent pre-RC formation, but the number, nature, and interplay of these mechanisms varies substantially between organisms.

	S. cerevisiae

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
Budding yeast initiates DNA replication at some 400 origins of DNA replication in every S phase. This organism is unique in that every known inhibitory mechanism is dependent on CDK activity (see Fig. 3). These mechanisms target each of the pre-RC components, sometimes in multiple ways. Cdc6 is inhibited at three levels. First, phosphorylation of Cdc6 marks it for ubiquitylation by the E3 ligase SCF^Cdc4, leading to proteasome-mediated destruction (Drury et al. 1997). Second, CDK inhibits Cdc6 transcription by blocking the nuclear import of the transcription factor, Swi5 (Moll et al. 1991). Finally, phosphorylation of Cdc6 at N-terminal CDK sites induces stable association with the mitotic CDK, Clb2–Cdc28, which blocks the licensing activity of Cdc6 (Mimura et al. 2004). The MCM2–7 complex is exported from the nucleus under the control of CDK phosphorylation (Labib et al. 1999; Nguyen et al. 2000; Liku et al. 2005), which also localizes Cdt1 to the cytoplasm during S phase, due to its association with MCM2–7 (Tanaka and Diffley 2002). ORC is inhibited by CDK phosphorylation of Orc2 and Orc6 (Nguyen et al. 2001). In addition, the S-phase cyclin Clb5 binds directly to an RXL motif in Orc6, but only after origins have fired. This interaction helps to inhibit the ability of ORC to assemble new pre-RCs (Weinberg et al. 1990; Wilmes et al. 2004). Given that Orc6–RXL mutations further increase rereplication in strains containing unphosphorylatable Orc2 and Orc6, it seems that binding of Clb5 to Orc6 is itself inhibitory to ORC activity, perhaps by creating a steric barrier that blocks binding of Cdc6 and/or Cdt1, or by locally increasing CDK activity. In summary, budding yeast cells appear to contain at least six independent, CDK-based strategies to prevent pre-RC formation during the S, G2, and M phases.

View larger version (38K): [in this window] [in a new window]   Figure 3. Species-specific pathways that prevent rereplication. The top of the figure shows the different stages of the eukaryotic cell cycle. For each species, each regulatory mechanism and the time in the cell cycle when it is active is represented by a horizontal bar. (Gray) Uncomfirmed pathways; (green) pathways that are directly regulated by CDK activity; (blue) PCNA-dependent Cdt1 proteolysis pathways; (yellow) Geminin.

The most difficult remaining question is what contribution any individual inhibitory mechanism makes in the context of the other mechanisms. Can some mechanisms be eliminated without any consequences to the cell? Although the CGH assay detects no rereplication in cells in which only the ORC complex is deregulated (Table 1, column 13), this assay is still not very sensitive, since a positive result requires rereplication at the same locus in a significant percentage of cells. Moreover, although these cells show normal viability, standard assays cannot detect small numbers of dying cells. Thus, only when rereplication is detectable in individual cells will it become possible to address this issue. If elimination of individual inhibitory mechanisms has detectable effects in single-cell assays, it will clearly demonstrate that each inhibitory mechanism makes measurable contributions toward the prevention of rereplication, even when many other mechanisms are operative. Such a scenario would explain how multiple inhibitory mechanisms are maintained during evolution.

Insulating licensing and initiation in S. cerevisiae

With respect to regulating DNA replication, the most challenging periods of the cell cycle occur at the transition points between low and high CDK activity, when origin firing and pre-RC formation could potentially overlap (for review, see Diffley 2004). Thus, at the M/G1 transition, when CDK activity drops, it is crucial to insure that conditions that allow origin firing decline before pre-RCs are allowed to reassemble (Fig. 1A). This "insulation" is achieved in yeast through a temporally ordered cascade of protein degradation. At the metaphase-to-anaphase transition, APC^Cdc20 ubiquitylates Clb5 and presumably Dbf4, eliminating the ability of cells to trigger replication initiation (Oshiro et al. 1999; Shirayama et al. 1999). Under these conditions, Clb2–Cdc28, which is poor at stimulating initiation, is still present and prevents pre-RC formation (Donaldson 2000). Subsequently, APC^Cdh1 triggers Clb2 destruction, allowing pre-RC formation. Thus, initiation becomes unfavorable before pre-RC assembly begins. Conversely, at the G1/S transition, it is crucial that the period of origin licensing ends before CDK activity rises, lest some origins initiate twice. To achieve this, G1 CDKs, which are not able to promote replication initiation, inhibit pre-RC formation via at least two mechanisms: Cdc6 proteolysis and MCM2–7 nuclear export (Labib et al. 1999; Drury et al. 2000; Perkins et al. 2001). Other organisms presumably also insulate the initiation and licensing periods from one another, and some details of how this might occur are discussed below.

In summary, all known mechanisms in budding yeast that prevent rereplication are CDK-dependent, and these various mechanisms are thought to combine multiplicatively to create a virtually insurmountable barrier to rereplication (Fig. 3).

	Schizosaccharoymces pombe

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
Like budding yeast, fission yeast replicates its genome from several hundred origins of replication spaced tens of kilobase pairs apart. S. pombe contains a single Cdk, called Cdc2. Cig1, Cig2, and Puc1 are S-phase cyclins, whereas Cdc13 is the mitotic cyclin. Rum1 inhibits the activity of Cdc2–Cdc13 (Correa-Bordes and Nurse 1995). As discussed above, the supreme role of CDKs in preventing rereplication was first discovered in fission yeast, where mutations in Cdc13, transient inhibition of Cdc2, or overexpression of Rum1 caused endocycles: multiple, discrete rounds of DNA replication with periodic origin licensing in the absence of mitosis. These observations suggest that in the absence of Cdc2–Cdc13, another Cdc2-dependent kinase stimulates DNA replication, and that this CDK undergoes periodic oscillations to allow repeated rounds of licensing and DNA replication. Cdc2–Cig2 is a good candidate because Cig2 overexpression in cells lacking Cdc13 prevents endoreduplication (Lopez Girona et al. 1998).

Early efforts to identify the targets of CDK inhibition focused on Cdc18, the fission yeast ortholog of Cdc6. As seen in budding yeast, Cdc18 is phosphorylated by Cdc2 in S phase, triggering ubiquitylation by the SCF^Pop1 ligase and destruction by the proteasome (Jallepalli et al. 1997; Kominami and Toda 1997). Initially, it was thought that Cdc18 might be the only target of CDK-dependent inhibition because high level Cdc18 overexpression, in particular alleles containing mutated CDK phosphorylation sites, causes rereplication (Nishitani and Nurse 1995; Jallepalli et al. 1997). However, a physiological level of nondegradable Cdc18 expression has no detectable effects on DNA replication in FACS assays, suggesting that other inhibitory mechanisms to prevent rereplication do exist (Nishitani and Nurse 1995; Muzi Falconi et al. 1996).

Like Cdc18, S. pombe Cdt1 is also regulated by proteolysis in the S and G2 phases (Nishitani et al. 2000; Gopalakrishnan et al. 2001). Cdt1 proteolysis requires a novel E3 ubiquitin ligase called Cul4–Ddb1^Cdt2 (Hu and Xiong 2006; Ralph et al. 2006). Interestingly, there is so far no evidence that this pathway is directly regulated by CDK, and experiments in other organisms show it is coupled to DNA replication (see below). Constitutive expression of Cdt1 alone is not sufficient to induce detectable rereplication, but when Cdt1 overexpression is combined with expression of nonphosphorylatable Cdc18, rereplication occurs, as seen by a gradual increase in ploidy (Nishitani et al. 2000; Gopalakrishnan et al. 2001; Yanow et al. 2001). Therefore, destruction of Cdt1 and Cdc18 represent overlapping mechanisms to prevent rereplication.

In addition to promoting Cdc18 destruction, Cdc2–Cdc13 inhibits pre-RC assembly by binding to the fission yeast Orc2 (called Orp2), at origins of replication (Wuarin et al. 2002). This mechanism is thought to locally antagonize pre-RC assembly either via Cdc18 and/or Orp2 phosphorylation (Vas et al. 2001) or by creating a steric barrier to pre-RC assembly. In either case, localization of Cdc2–Cdc13 to origins is specific to G2 and M phase, and origin localization of Cdc2 is not conferred by S-phase cyclins. Interestingly, ablation of Cdc2–Cdc13 binding to Orp2 does not cause rereplication, but endoreduplication (Wuarin et al. 2002). Thus, association of Cdc13–Cdc2 with Orp2 is not essential to prevent origin refiring within S phase. Rather, this mechanism ensures that within G2 and M phase, further rounds of pre-RC assembly are blocked until the cell has entered anaphase.

In summary, S. pombe uses at least two strategies to prevent replication within S phase (Cdt1 and Cdc18 proteolysis), as well as a third mechanism to prevent relicensing in G2 and M (ORC inactivation by Cdc2–Cdc13) (Fig. 3). Since MCM2–7 is constitutively nuclear, S. pombe may not inhibit this pre-RC component.

	X. laevis

Top Abstract The two-state model for... Step one: licensing origins... Step two: initiation of... Anaphase-promoting complex... Rereplication, failed mitosis,... Redundancy and the regulation... Strategies to prevent... S. cerevisiae Schizosaccharoymces pombe X. laevis Mammals Caenorhabditis elegans Flies Endocycles Consequences of rereplication Rereplication and disease Conclusions and outlook Acknowledgments References

 
The early embryonic cells cycles of the frog X. laevis take 30 min and consist of alternating S and M phases without intervening gap phases (for review, see Blow 2001). DNA replication initiates in a sequence-independent fashion at 300,000 origins spaced 10 kb apart. Replication initiation is dependent on Cdk2–Cyclin E, mitotic entry is driven by Cdk1–Cyclin B, and mitotic exit is promoted by APC^Cdc20. Two unique features of these cell cycles are noteworthy. First, these cells do not express APC^Cdh1, explaining the absence of a G1 period (Lorca et al. 1998). Second, the activity of key cell cycle regulators fluctuates largely as a result of subcellular localization rather than expression level. Thus, Cdk2–Cyclin E levels are constant throughout the cell cycle (Hua et al. 1997). However, in mitosis, when there is no nuclear envelope, the Cdk2–Cyclin E concentration surrounding chromatin is low. Starting at telophase, Cdk2–Cyclin E is imported into nuclei, causing a dramatic rise in its nuclear concentration that triggers initiation. Importantly, extracts prepared from unfertilized Xenopus eggs undergo precisely one round of DNA replication per in vitro cell cycle, making this a powerful tool to understand the regulation of DNA replication.

Geminin

Geminin was initially discovered in Xenopus egg extracts in a screen for APC^Cdc20 substrates, and it was immediately recognized as an inhibitor of MCM2–7 loading (McGarry and Kirschner 1998). Present in all metazoans but apparently not in yeast, Geminin inhibits pre-RC assembly by sequestering Cdt1 in an inactive complex that is unable to interact with or recruit MCM2–7 (Wohlschlegel et al. 2000; Tada et al. 2001; Cook et al. 2004; Lee et al. 2004; Ferenbach et al. 2005; Lutzmann et al. 2006). Interestingly, Geminin does not inhibit the interaction of Cdt1 with origins of DNA replication (Gillespie et al. 2001). Crystal structures and mutational analyses reveal that Geminin assembles into a homodimer with a central coiled-coil domain that interacts extensively with Cdt1, and thereby probably creates a steric barrier that prevents association of Cdt1 with MCM2–7 (Lee et al. 2004; Saxena et al. 2004).

In Xenopus egg extracts, Geminin is inactivated by APC^Cdc20 at the metaphase–anaphase transition (McGarry and Kirschner 1998). Together with the destruction of mitotic cyclins, this event inaugurates the licensing period. Interestingly, only about half the endogenous Geminin is degraded in anaphase; the remainder is inactivated via an unknown mechanism that involves Geminin ubiquitylation (Li and Blow 2004). Geminin is reactivated when licensing ends in telophase, indicating that the licensing period during these cell cycles lasts only a few minutes. The mechanism of reactivation is not understood, but it is dependent on nuclear import of Geminin, after which the binding of Geminin to Cdt1 increases dramatically (Hodgson et al. 2002; Arias and Walter 2005; Lutzmann et al. 2006).

The properties of the Geminin that persists during the licensing period are controversial. The conventional view is that inactivated Geminin is unable to bind Cdt1 and therefore does not prevent licensing (Hodgson et al. 2002). However, a recent study suggests that Geminin and Cdt1 form a complex that is competent for licensing (Lutzmann et al. 2006). In this view, Geminin only inhibits Cdt1 activity when a critical Geminin:Cdt1 ratio is achieved, which occurs after Geminin is reactivated in telophase. The fact that Xenopus Geminin is regulated post-translationally is probably crucial to allow it to switch rapidly between functional states, thereby accommodating the rapid embryonic cell cycles. It is unclear whether somatic cells contain similar populations of Geminin that are unable to inhibit the function of Cdt1.

The ability of Geminin to inhibit Cdt1 is apparently independent of CDK status (Ballabeni et al. 2004; Sugimoto et al. 2004; Li and Blow 2005). As such, the inhibition of Cdt1 by Geminin represents the first known CDK-independent means to prevent pre-RC assembly. Notably, immunodepletion of Geminin from Xenopus egg extracts was reported to induce no detectable rereplication (McGarry and Kirschner 1998), suggesting the existence of additional mechanisms to prevent rereplication (see next section).

PCNA-dependent and Cul4–Ddb1^Cdt2-dependent Cdt1 proteolysis

Cdt1 is destroyed in the S phase of all metazoan cells (Nishitani et al. 2001; Zhong et al. 2003; Thomer et al. 2004; Arias and Walter 2005), and it has recently emerged that its proteolysis is intimately linked to DNA replication (Fig. 4A). Thus, in Xenopus egg extracts, it was first discovered that Cdt1 is ubiquitylated on chromatin in a manner that depends on the initiation of DNA replication (Arias and Walter 2005). Subsequent analysis showed that Cdt1 destruction depends on its binding to PCNA (Arias and Walter 2006). Xenopus Cdt1 contains a PCNA-interacting protein (PIP) box at its N terminus (shown as a red box in Fig. 4A). Mutation of the PIP box abolishes Cdt1 binding to PCNA, its ubiquitylation on chromatin, and its destruction in S phase. Interestingly, PCNA-dependent Cdt1 ubiquitylation occurs exclusively on chromatin. Therefore, it appears that Cdt1 can only interact with chromatin-bound PCNA, a feature of the proteolysis mechanism that prevents Cdt1 destruction in G1 phase. Because it participates in the synthesis of every Okazaki fragment, PCNA accumulates to high levels on chromatin during S phase (Arias and Walter 2006), making it a well-suited trigger for rapid Cdt1 destruction. Importantly, all metazoan organisms contain a PIP box at the extreme N terminus of Cdt1 (Arias and Walter 2006; Senga et al. 2006), and current evidence indicates that PCNA-dependent Cdt1 destruction is highly conserved among these organisms (see below).

View larger version (57K): [in this window] [in a new window]   Figure 4. Cdt1 proteolysis pathways. The two major pathways that target Cdt1 for destruction are shown. The PIP box of Cdt1 is indicated in red. (A) Cul4–Ddb1Cdt2-dependent Cdt1 destruction occurs in two steps. First, Cdt1 docks onto PCNA at DNA replication forks or sites of DNA damage. Second, Cdt2 interacts with the complex of Cdt1 and PCNA and ubiquitylates Cdt1. (B) SCFSkp2-dependent Cdt1 destruction also occurs in two steps. First, CDK phosphorylates Thr 29 (in humans). Second SCFSkp2 binds to the phoshporylated T29 via Skp2 and ubiquitin tranfer occurs.

What is the division of labor between Geminin and Cdt1 destruction in preventing rereplication? Blocking proteolysis of Cdt1 by itself yields no detectable rereplication (Arias and Walter 2005, 2006; Li and Blow 2005; Maiorano et al. 2005; Yoshida et al. 2005). Similarly, in the absence of Geminin, rereplication is not detectable (McGarry 2002; Arias and Walter 2005) or very inefficient (Li and Blow 2005; Yoshida et al. 2005; Kerns et al. 2006). However, when both mechanisms are neutralized, substantial rereplication occurs (Li and Blow 2005; Maiorano et al. 2005; Yoshida et al. 2005; Arias and Walter 2006). Therefore, it appears that Geminin and Cul4–Ddb1^Cdt2-dependent Cdt1 destruction are each sufficient to prevent the large majority of rereplication in interphase egg extracts.