Introduction

Checkpoints are mechanisms that establish dependence relationships between biochemically unrelated cellular processes. The temporal order of many critical cell cycle events must be strictly maintained to ensure cell survival and integrity. A simple example is that of genome duplication which must be completed before cell division. The relationship between these processes is controlled by the S-phase checkpoint. After S-phase, the topoisomerase II-dependent checkpoint ensures that the topology of the newly replicated DNA has been correctly organized before cells begin mitosis. During mitosis itself, distinct checkpoints monitor mitotic spindle assembly, preventing the onset of chromosome segregation until all the chromosomes are correctly aligned on the mitotic spindle, and prevent exit from mitosis until anaphase chromosome segregation has been completed. In this chapter, we discuss these checkpoint control systems (Fig. 1).

Figure 1

DNA damage-independent checkpoints. A summary of the five checkpoints that are discussed in detail (see magnifying glass). In mammals the G2/M transition is regulated by at least 2 non-DNA damage checkpoint pathways (1 and 2). These prevent the initiation (more...)

An elegant way to define checkpoint pathways has been by analysis of loss-of-function mutants in the genetically manipulatable yeast systems. For example, the S-phase checkpoint was described in budding yeast by the isolation of mutants that initiate mitosis despite a replication block enforced by the ribonucleotide reductase inhibitor hydroxyurea (HU). Other checkpoint systems were originally described in mammalian cells. Indeed, the existence of checkpoint controls had been inferred from mammalian cell-fusion studies earlier than the genetic analyses performed in yeast (Fig. 2).¹ More recently, mammalian checkpoints have been investigated by demonstrating that some checkpoint arrests can be overridden by drugs such as caffeine.^2-9 Caffeine inhibits the kinase ATM, a key component of eukaryotic checkpoint pathways.^10-13 Although mammalian cells are less amenable to genetic studies than are yeast, they have proven to be important for the study of checkpoint biology.

Figure 2

Premature chromosome condensation (PCC). Interphase cells can be fused with mitotic cells by Sendai virus treatment. This induces PCC, suggesting that interphase checkpoints might normally inhibit the onset of chromosome condensation. Photomicrographs (more...)

Our descriptions of checkpoint controls use a commonly adopted format which divides the pathways into (1) the sensor, (2) the transducer and (3) the target. Checkpoints do not necessarily follow simple linear pathways, however. Many of the sensor and transducer components are likely to be assembled into large complexes. Still, this nomenclature allows for a framework to be drawn up, on which the details can be built. The sensor components are those that monitor completion of the relevant process, for example DNA replication. The transducer transmits the signal from the sensor to the target of the checkpoint. It is the activity of the target that controls cell cycle progression.

Budding Yeast Versus Higher Eukaryotes

Since checkpoint pathways in both budding yeast and higher eukaryotes will be discussed in this chapter, it is important to describe a fundamental difference in budding yeast cell cycle organization that sets it apart from other species. In budding yeast, checkpoints promote the activity of anaphase inhibitors, whereas in most eukaryotes, checkpoints inhibit the activity of the mitotic kinase (cyclin/Cdk), required for passage through the G2/M transition. A need for distinct modes of control is related to differences in the spindle assembly pathway. In many eukaryotes, including mammals, the mitotic spindle does not assemble until mitosis. However, budding yeast spindles assembled during S-phase; checkpoints must inhibit spindle elongation even while DNA is being replicated. In addition, sister chromatid cohesion, established during DNA replication, must be maintained until the onset of anaphase. In budding yeast, an inhibitor of anaphase, Pds1, can perform both of these tasks.^14-18 Before anaphase, Pds1 binds to protease Esp1 and thereby inhibits the anaphase-promoting activity of Esp1.¹⁸ During an unperturbed cell cycle, Pds1 becomes poly-ubiquitinated at the metaphase to anaphase transition by a multi-subunit enzyme complex known as the APC (Anaphase Promoting Complex); the modified forms are recognized and degraded by 26S proteasomes.¹⁶ Once released from Pds1, Esp1 induces cleavage of Scc1, a cohesin required to maintain cohesion between sister chromatids.^18-21 Concurrently with loss of sister cohesion, Esp1 induces spindle elongation.²² Not surprisingly, Pds1 is a major target of checkpoints controlling anaphase onset. Vertebrate proteins named Securins, that are at least partial functional homologues of Pds1, have been identified,²³ making the study of checkpoints in budding yeast highly relevant. Moreover, budding yeast and higher eukaryotes employ a common strategy for controlling exit from mitosis. In this case, regulation of cyclin/Cdk activity appears to be a universally adopted mode of control.

S-Phase Checkpoint

The S-phase checkpoint ensures that the onset of mitosis is dependent on the completion of DNA replication.^24-26 Since little is known about S-phase checkpoint control in mammals, the components of this pathway in budding yeast will be described (Fig. 3). As mentioned above, budding yeast cells initiate DNA replication and mitotic spindle formation at a common cell cycle point, early in S-phase. To prevent the generation of aneuploid daughter cells that are inviable, it is essential that the mitotic spindle does not elongate before DNA replication has been completed. The order of these two processes is normally maintained by a timing mechanism rather than a checkpoint control: DNA replication takes only 20–30 minutes and spindle formation takes around 60 minutes; thus, spindle assembly is not completed before genome replication. This example illustrates how the temporal order of two events can be maintained independently of checkpoint controls, i.e. if the processes have a common starting point and each require a differing minimum amount of time for their completion.

Figure 3

S-phase checkpoint. Linear pathways are drawn for simplicity though more complex interactions between the checkpoint components are likely. In budding yeast, a signal generated by replication forks is transmitted by kinases Mec1 and Rad53. Rad53 activation (more...)

A checkpoint pathway does exist, however, to ensure that the dependence between spindle elongation and DNA replication is always maintained. If DNA replication is inhibited with HU,²⁷ the cells arrest with fully assembled short G2 spindles. After removal of the HU, spindle elongation is delayed until replication is complete. The S-phase checkpoint does not only control the mitotic spindle, however. All eukaryotes establish sister cohesion during DNA replication, and it must be maintained until the onset of anaphase.^20,21 Maintenance of sister chromatid cohesion is of great importance to mammals and yeast alike and is a function of the S-phase checkpoint. At least in yeast, cohesion is established at some loci very early in S-phase and must therefore be maintained for the remainder of the S-phase period as well as during G2 and until the moment of anaphase onset. The homologs of yeast S-phase checkpoint components are therefore likely to be important regulators of mammalian sister chromatid cohesion.

To define the budding yeast S-phase checkpoint, loss-of-function mutations causing sensitivity to HU were identified. The proteins encoded by these genes were determined to have S-phase checkpoint functions by showing that the loss-of-function mutations allowed entry into mitosis when DNA replication was blocked with HU. Thus the S-phase checkpoint is defined as that which restrains entry into mitosis when replication is blocked. However, kinetic analyses of various checkpoint mutants, grown in the presence of a concentration of HU that allows replication to proceed, but more slowly than in an unperturbed cell cycle, have revealed genetically distinct S-phase checkpoint systems (see below).

To monitor ongoing DNA replication, cells seem to have replication sensors that reside at replication forks. In budding yeast, the putative sensor components include Pol2, Rfc5, Dpb11, Drc1 and Sgs1.^28-32 POL2 encodes the replicative DNA polymerase, Pole, and Rfc5 is a replication factor C subunit involved in recruiting polymerases to replication forks. Dpb11 is also required for DNA replication; it can bind to Pole and is thought to help recruit Pola-primase complexes to ARS sequences at replication origins.³³ Dpb11 also binds to Drc1, which is itself essential for DNA replication.³¹ Together with Srs2, Sgs1 has a redundant but essential role in DNA replication.³⁴

Evidently, the sensor proteins also play important roles in DNA replication itself, and with hindsight it might seem elementary that components of the replication fork machinery are involved in generating the checkpoint signal. For each of these components, it was important to know that their checkpoint functions could be distinguished from their roles in DNA replication. This is the case because the S-phase checkpoint cannot be activated until DNA replication has begun,³⁵ a fact illustrated by the phenotype of cells carrying a heat-inducible cdc45 degron mutant.³⁶ Cdc45 binds to replication origins before S-phase in budding yeast and is required for origin firing. The cdc45 degron mutant is rapidly degraded at the restrictive temperature. When degradation was induced in G1 of the cell cycle, replication origins could not fire, and the cells entered mitosis without replicating any DNA. When the temperature shift was performed within S-phase, DNA replication was immediately inhibited because Cdc45 is also needed for elongation of replication forks, but in this case mitotic progression was inhibited. Thus, the S-phase checkpoint signal requires the presence of replication forks that have fired, and Cdc45 is not a component of the checkpoint response.

Mec1 and Rad53 kinases are traditionally described as components of the signal transduction element of the S-phase checkpoint.^24,25,37 When replication is perturbed, these checkpoint kinases are activated in a manner dependent on the sensor components. Exactly how Rad53 and Mec1 activation occurs is not known, but several physical interactions have been identified that may represent key steps. Sgs1 was found to co-localize with Rad53 in discrete nuclear foci during S-phase. Intriguingly, Sgs1 is reported to interact with the FHA domain of Rad53,³⁸ the domain required for the formation of the Rad53-Rad9 complex, required for DNA damage checkpoint signaling.³⁹ Rad53-Sgs1 association may have revealed an Sgs1-dependent loading of Rad53 onto specific chromatin regions that might be involved in monitoring replication. That Sgs1 is involved in S-phase checkpoint control,³² adds weight to this model. But, the S-phase checkpoint defect of sgs1 mutants is rather weak, not nearly as substantial as other S-phase checkpoint mutants. This suggests that Sgs1 has a redundant checkpoint function, perhaps with another helicase such as Srs2.

Another activator of signal transduction may be Ddc2 (also known as Lcd1), a component that physically associates with Mec1 and is phosphorylated by Mec1.^40,41 Phosphorylated Ddc2 is present in unperturbed S-phase cells and in HU-treated cells, and Ddc2 is required for cell cycle arrest in the presence of HU. Phosphorylation and activation of Rad53 in response to replication arrest is Ddc2-dependent. Therefore, Ddc2 appears to mediate between Mec1 and Rad53 in response to ongoing DNA replication and when fork progression is blocked. It remains to be tested whether the association of Ddc2 with Mec1, or the phosphorylation of Ddc2 by Mec1, depends on the sensor components, and how these events are regulated.

In response to replication inhibition, Mec1 and Rad53 enforce cell cycle arrest by blocking Pds1 degradation. In addition, these kinases induce transcription of genes involved in DNA repair and that help deal with the perturbed replication process.²⁴ This safety system most likely protects stalled replication forks, allowing them to be re-initiated when conditions have improved. The transcription pathway depends on Rad53-dependent phosphorylation of the kinase Dun1 (damage uninducible).^24,42 Activation of Dun1, in response to DNA damage or DNA replication blocks, induces transcription of genes that promote efficient DNA repair.²⁴ This transcription response is partially initiated by Crt1 hyperphosphorylation.⁴² Crt1 represses transcription of DNA damage-inducible genes by binding to their promoter regions; binding is prevented by hyperphosphorylation. Activation of a transcription program clearly has the potential to enforce a wide range of Mec1- and Rad53-dependent functions, and a growing literature has made clear that Mec1 and Rad53 are involved in numerous cellular processes. For example, Mec1 and Rad53 inhibit the firing of late replication origins during early S-phase. Eukaryotic cells replicate their genomes by initiating DNA synthesis from multiple replication origins. Some fire early in S-phase, others are initiated part way through S-phase. When cells are arrested in early S-phase with HU, late firing origins are kept dormant by a dominant process that requires the Rad53-Mec1 pathway.⁴³ Mec1 and Rad53 are also involved in the regulation of telomere length and in transcriptional silencing at telomeres.^44-46

It is not clear, however, whether the Mec1/Rad53/Dun1-dependent transcriptional response contributes to cell cycle arrest in the presence of HU, since dun1 null mutants are not S-phase checkpoint defective. In agreement with this, the cell cycle checkpoint defects of mec1 and rad53 mutants are somewhat different. Both mutants elongate their mitotic spindles when DNA replication is blocked with HU, so it seems that no checkpoint response remains in these cells. However, rad53 mutants delay in anaphase, while mec1 mutants exit mitosis. Thus some aspects of mitotic progression are inhibited in rad53 mutants. Light was shed on the basis of this difference by analysis of pds1 mutants, revealing that there are several S-phase checkpoint targets. Pds1 is not an essential target in early S-phase because pds1 mutants can inhibit spindle elongation when replication is blocked with HU in early S. However, kinetic analyses determined that, part way through S-phase, a critical point is reached where Pds1 becomes essential: pds1 mutants elongate spindles and lose sister chromatid cohesion when roughly 2/3 of the genome has been replicated.⁴⁷ These experiments were performed in the presence of a concentration of HU that does not fully block replication, but instead, slows the rate of DNA replication. In these experiments, mec1 or rad53 mutant cells began anaphase when very little DNA had been replicated (as is the case when replication is blocked). Therefore, a Pds1-independent system restrains spindle elongation in early S-phase, but later in S-phase, Pds1 is required. A reasonable prediction is that Pds1 and Rad53 function downstream of Mec1 in the context of S-phase checkpoint control, and that these pathways run in parallel, and are temporally regulated; one necessary in early S-phase, the other part way through S-phase. Presumably, Mec1 controls Pds1 stability in late S-phase. Several details remain unresolved, however. For example, the fact that pds1 null mutants are able to restrain spindle elongation and prevent premature loss of sister chromatid cohesion in early S-phase necessitates a novel Mec1/Rad53 target at that point in the cell cycle.

An explanation for the duality of S-phase checkpoint control in budding yeast is the linkage of spindle elongation with regulation of sister cohesion. Sister cohesion is established during DNA replication and must be maintained until the onset of anaphase.^20,21 Once cohesion is established, part way through S-phase,⁴⁸ checkpoint control of anaphase must coordinate release of cohesion with spindle elongation. Early in S-phase, prior to replication of critical cohesion regions and concomitant establishment of cohesion, spindle elongation might be regulated independently of cohesion. Therefore, the switch in the mode of checkpoint control from the Mec1-Rad53 pathway to the Mec1-Pds1 pathway may be controlled by the establishment of sister chromatid cohesion.

It remains to be determined how Pds1 levels are controlled in late S-phase when DNA replication is perturbed. Recent evidence has linked two yeast genes to regulation of Pds1 in this context.⁴⁹ Rad23 or Ddi1 overproduction was found to rescue the sensitivity of pds1 mutant cells to HU. Rad23 is a nucleotide excision repair protein, but recent studies suggest a novel role of Rad23 in ubiquitin-dependent proteolysis.⁵⁰ Rad23 binds to mono- or di-ubiquitinated proteins but cannot bind when the ubiquitin chains have been elongated. Crucially, Rad23 blocks extension of the ubiquitin chains. For most ubiquitinated proteins that are targeted for degradation, ubiquitin chain elongation is critical for efficient recognition by the 26S proteasome. Therefore, Rad23 might have an important function in preventing or delaying the degradation of proteasome targets. Although this mechanism has not been tested directly in the context of S-phase checkpoint control, overexpression of Rad23 is able to stabilize a mutant pds1 protein, suggesting that Rad23 might play a role in S-phase checkpoint signaling.⁴⁹ A role of Rad23 in checkpoint signaling may be utilized by virally expressed proteins. The HIV-1 encoded protein Vpr has been shown to bind to the C-terminal UBA (Ubiquitin associated domain) of human Rad23 (HHR23A).^51-53 This interaction is needed for one of the cellular functions of Vpr, the ability of Vpr to induce G2 cell cycle arrest, which allows time for viral replication. It seems plausible that Vpr mimics an endogenous cellular checkpoint response that involves binding of the Rad23 UBA to an unknown protein, inducing G2 arrest.

Mammalian cells also need an S-phase checkpoint. The initiation of mitosis must be prevented during S-phase, and chromatid cohesion must be maintained. Mammalian Sgs1 homologs are clearly important for S-phase regulation.³⁸ Sgs1 is a budding yeast member of the Escherichia coli recQ helicase family, and sgs1 mutants are genomically unstable.⁵⁴ Mammalian recQ helicase family members include WRN (mutated in Werner's syndrome patients)⁵⁵ and BLM (mutated in Bloom's syndrome patients).⁵⁶ Bloom's syndrome is characterized by genomic instability and a high incidence of cancer, whereas Werner's syndrome causes premature ageing. The BLM protein was recently identified as a component of a large complex that includes tumor suppressor proteins, DNA repair and checkpoint proteins that localize to nuclear foci when cells are treated with HU.⁵⁷ Indeed, cultured cells from Bloom's syndrome patients have S-phase defects, but it is not clear whether these abnormalities include checkpoint abrogation. In general, there are mammalian homologues of all the budding yeast checkpoint proteins, but their potential roles in S-phase checkpoint control have not been thoroughly investigated.

The Mec1 homologs are ATM and ATR.^58,59 ATM, the gene mutated in ataxia telangiectasia patients who have an increased incidence of cancer, is a nuclear protein kinase. ATR (ataxia telangiectasia and rad3 related) is also a protein kinase and is structurally more homologous to Mec1 than is ATM. Both ATM and ATR are clearly involved in DNA damage checkpoint signaling,^60,61 but do not seem to be required for preventing the onset of mitosis during S-phase. Whether these proteins have roles in regulating cohesion has not been addressed. In the context of the DNA damage checkpoint, the tumor suppressor proteins p53 and Brca1 (breast cancer gene 1) seem to be targets of ATM/ATR, but again there is little evidence for roles in S-phase.^62-68 Brca1 is, however, phosphorylated by ATR in response to HU treatment.⁶⁹

The mammalian Rad53 homologue, kinase Chk2 (Checkpoint kinase),⁷⁰ and the mammalian homologue of budding and fission yeast Chk1 (also named Chk1), are required for the ATM-dependent DNA damage checkpoint.^71,72 After γ-irradiation, mammalian Chk2 phosphorylation (on Thr-68, within the serine/threonine cluster domain of Chk2) and activation is ATM-dependent.⁷³ Chk2 kinase also becomes phosphorylated and activated upon HU treatment, but in an ATM-independent manner, and not on Thr-68.^70,73,74 If the HU-induced phosphorylation is relevant for S-phase checkpoint control, it might be that the mammalian S-phase checkpoint operates by a kinase distinct from ATM.

Chk1 is not needed for S-phase checkpoint control in budding yeast but does seem to be required in some higher eukaryotes. Xenopus Chk1 is activated in post-MBT (mid-blastula transition) embryonic cells treated with HU.⁷⁵ Similarly, there is good evidence for a role of Drosophila Chk1 (named Grapes) in coordinating embryonic DNA replication with the onset of mitosis.^76,77 In Xenopus egg extracts, immunodepletion of Chk1 impairs an ability to delay cell cycle progression in response to replication blocks.⁷⁸ Immunodepletion of ATR has the same effect since Chk1 activity depends on phosphorylation by ATR when unreplicated DNA is present.⁷⁹ Human ATR has been implicated in Chk1 phosphorylation in response to HU treatment, but it is not known if cells lacking Chk1 or ATR have defective S-phase checkpoint controls.⁸⁰ In Xenopus, Chk1 activation also depends on a protein named Claspin which has a close human homolog. Xenopus egg extracts depleted of Claspin are S-phase checkpoint deficient.⁸¹

Both Chk1 and Chk2 can phosphorylate Cdc25C on Ser-216 in humans and it is thought that this phosphorylation prevents Cdc25C from activating the mitotic kinase, cyclinB1/Cdc2.^70,71 Cdc25C is a protein phosphatase that promotes entry into mitosis by dephosphorylating Cdc2. This phosphorylated residue creates a binding site for a 14-3-3 protein, resulting in Cdc25C inhibition.⁷² Interestingly, expression of a mutant Cdc25C that cannot be phosphorylated on Ser-216 induces mitosis in the presence of unreplicated DNA, suggesting that this pathways may be important for S-phase checkpoint control.⁷²

Topoisomerase II-Dependent Checkpoint

DNA topoisomerase II (topo II) is required for chromosome condensation and segregation in eukaryotes.^3,82-87 Although these are mitotic processes, their successful completion depends partly on topo II activity during DNA replication and in G2 phase (Fig. 4). Chromosome replication creates two identical sister DNA molecules that are knotted together (catenated). Topo II removes the catenations; in higher eukaryotes, the majority must be resolved before entry into mitosis to allow accurate chromosome condensation.^3,88 A G2 checkpoint ensures that DNA catenations have been sufficiently resolved before mammalian cells enter mitosis.³

Figure 4

Chromosome dynamics mediated by topoisomerase II (topo II). Topo II decatenation reactions are needed for various mitotic steps in mammalian cells. Before mitosis, chromosome individualization is promoted by the resolution of non-replicative catenations (more...)

It had been known for some time that topo II inhibitors block or delay mammalian cell cycle progression in G2, but the inhibitors used were also known to cause DNA damage; it was assumed that the G2 arrest was due to activation of the DNA damage checkpoint. That cells also need to monitor topo II activity in G2 was an attractive hypothesis, however, and the characterization of novel topo II inhibitors that do not damage DNA allowed this theory to be tested. A variety of assays that measured DNA damage were used to assess the effects of bisdioxopiperazine topo II inhibitors on mammalian cells and on isolated DNA. Bisdioxopiperazines were found not to induce DNA breaks in vivo or in vitro³ (and Refs. therein) but did block mammalian cells in G2. In these studies, entry into mitosis was assessed based on the onset of chromosome condensation. Yet, topo II is needed for chromosome condensation, albeit a requirement at late steps in the process.^3,88 It was therefore necessary to prove that cells were physically capable of initiating chromosome condensation when topo II activity was absent. Such evidence could not be sought by isolating loss-of-function budding or fission yeast mutants, since the topo II-dependent checkpoint seems to be absent in yeast.^85,89 Therefore the checkpoint was first described in mammals, by demonstrating checkpoint bypass induced with caffeine or kinase and phosphatase inhibitors.³ Cells treated with ICRF-193, the most potent of the bisdioxopiperazines, could be forced into mitosis with caffeine; the cells began to condense chromosomes without delay. Fully condensed chromosomes were not formed, consistent with the essential role of topo II late in the condensation process. Thus, the topo II-dependent checkpoint prevents the onset of chromosome condensation, a process which the cells can begin, but cannot complete in the absence of topo II activity.

Although the evidence for this checkpoint in mammals is substantial, its absence in yeast has prevented the checkpoint components from being rapidly identified. Indeed, it is not known whether topo II levels are sensed directly, or if physical structures within chromosomes, such as catenations (Fig. 5), are monitored. Some data suggest the latter is more likely. Replicative catenations are introduced between daughter DNA duplexes during S-phase. Disentangling daughter duplexes is of crucial importance since they otherwise could not separate and segregate during mitosis. Most replicative catenations are resolved in G2, when cellular topo II activity increases, but since the decatenation reaction is reversible, topo II activity inevitably promotes some catenation. This generates non-replicative catenations, that can involve distant regions of chromatin, and can join different chromosomes together.^90,91 Cells inhibited for topo II activity late in G2 and forced to enter mitosis with caffeine, have striking chromosome aberrations caused by persistent non-replicative catenations that join chromosomes together and create W-figures within individual chromosomes (Fig. 4 and Fig. 5).⁹² Circumstantial evidence suggests that the removal of non-replicative catenations in G2, the process that promotes chromosome individualization, may be monitored by the topo II-dependent checkpoint.⁹²

Figure 5

Location of putative topoisomerase II-dependent checkpoint sensors. The cartoons depict a model of chromosome structure showing loops of chromatin attached to a chromosome core or scaffold, and the location of non-replicative and long-lived replicative (more...)

Another question is what is the nature of the topo II-dependent checkpoint sensor? It might be that sensors bind to sites of DNA catenation. Though purely speculative, there is a precedent for such a proposal, that a signaling cascade might be activated by protein complexes at sites of DNA crossover. In bacteria, stable maintenance of the natural multicopy plasmid CoIE1 requires a cer sequence element (Fig. 6). cer is necessary for recombination that converts unstable plasmid multimers to monomers.^93,94 The expression of Rcd, a transcript encoded from within cer, is specifically expressed in cells containing multimers. Rcd1 enforces a cell cycle checkpoint that inhibits cell division when multimers are present,⁹⁵ thus allowing time for site-specific recombination to occur. An interesting observation is that the Rcd1 promoter resides within the cer sequence, and that the topology of cer is likely to be altered in multimers that assemble recombination complexes at the cer sites.⁹⁶ This difference in topology might influence Rcd1 transcription, providing an elegant mechanism to activate the checkpoint in the presence of multimers. The parallel between this phenomenon in bacteria and the topo II-dependent checkpoint signal generated by persistent DNA catenations in mammalian cells is remarkable.

Figure 6

A model for the bacterial Rcd checkpoint. Rcd imposes a cell cycle arrest before cell division that allows time for plasmid multimers to be recombined into stable monomers. Rcd is encoded from within the cer element of the recombination site. Transcription (more...)

The target of the topo II-dependent checkpoint is presumed to be mitotic cyclin/Cdk activity: recent work has shown that a topo II-dependent checkpoint exists in plant cells that can be overridden by overexpressing a mitotic cyclin (J.F.G.A., unpublished data). How the mitotic kinase is regulated in response to topo II-dependent checkpoint activation is not known, although some data give clues as to the upstream components of the pathway. The topo II inhibitor genistein arrests mammalian cells in G2 by activating Chk2 kinase, which in turn leads to the inhibition of Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1.⁹⁷ Activation of Chk2 occurs very efficiently at genistein doses that inhibit topo II but cause minimal DNA damage compared to other topo II inhibitors such as etoposide. In the case of genistein, ATR might be the kinase upstream of Chk2, since caffeine overrides the G2 arrest whereas Wartmannin does not. In contrast to ATM, which is inhibited by caffeine and Wartmannin, ATR is only efficiently inhibited by caffeine.⁹⁷ The topo II-dependent checkpoint and DNA damage checkpoint might be regulated primarily by ATR and ATM, respectively. Although these checkpoints are distinct, the possibility remains that they are closely linked pathways. One way to address this issue will be to test whether other components of the DNA damage checkpoint, such as Chk1, p53 and 14-3-3s, are activated in the context of ICRF-193-induced G2 arrest.

Checkpoint Control in Prophase

A recent study identified a novel mammalian checkpoint protein Chfr (Checkpoint with FHA and Ring finger) apparently acting to slow chromosomal condensation in prophase and prometaphase when microtubule polymerization is perturbed.⁹⁸ In a cohort of 8 human tumor cell lines, three were identified that failed to express Chfr at the transcriptional level, although this was not due to loss of both gene copies. Furthermore, a mutation was identified in a fourth cell line leading to loss of Chfr function. In tumor cell lines lacking Chfr function, mitotic chromosome condensation occurred at the same rate in the presence or absence of nocodazole (or taxol). In cells with functional Chfr, or cells lacking Chfr but transiently transfected with a functional copy, chromosome condensation occurred at a reduced rate in nocodazole-treated cells, relative to cell cycle progression from G2 to metaphase based on the accumulation of cyclin/Cdk activity and prophase separation of centrosomes. Examination of nuclear morphology and DNA content following 48 hours of microtubule depolymerizing treatment demonstrated that Chfr-defective cells undergo aberrant mitosis, implicating this checkpoint in chromosome instability. While there is no definitive yeast homolog for Chfr, two S. cerevisiae open reading frames and one S. pombe gene, defective in mitotic arrest (Dma1),⁹⁹ appear closely related. Clearly, study of a larger cohort of tumor cell lines and further mechanistic studies need to be performed to fully assess the import of this checkpoint in tumorigenesis. These may also substantiate the authors claims that Chfr is inactivated more frequently than ‘all known spindle checkpoint proteins combined’.⁹⁸

Spindle Assembly Checkpoint

The spindle assembly checkpoint is an example of how a combination of yeast genetics and cell biology in higher eukaryotes has rapidly expanded our knowledge of a biological system.^26,100-104 Eukaryotic cells arrest in metaphase when microtubule polymerization is disturbed. Higher eukaryotic cells, with normal mitotic spindles, also arrest if chromosomes fail to become bioriented on the spindle and have not congressed to the metaphase plate.¹⁰⁵ Other defects such as spindle pole body (SPB), kinetochore and centromere abnormalities also activate the checkpoint,¹⁰⁰ and it is known that incorrect spindle orientation (relative to the cell axis) can delay the onset of anaphase.¹⁰⁶ Therefore, the spindle assembly checkpoint monitors the process of bipolar attachment of all the chromosomes to the mitotic spindle and ensures that the spindle is correctly positioned.

Chromosomes become bioriented by amphitelic attachment of their kinetochores to spindle microtubules.^100,107 The process of chromosome capture by the spindle occurs more or less randomly;¹⁰⁷ within the same species and cell type, it is accomplished quickly in some cells, but takes much longer in others.¹⁰⁵ Therefore, in animals and in yeast, the checkpoint is needed in every cell cycle.^108-110 However, biorientation of the chromosomes on the mitotic spindle forms a stable structure,^107,111 thus the correct alignment of chromosomes, creating the metaphase plate, is favored. Once the last chromosome becomes bioriented, the spindle assembly checkpoint signal diminishes and anaphase is initiated in a highly regimented manner.¹⁰⁵

At least one facet of the checkpoint signal emanates from kinetochores that have not attached to the spindle.¹¹² In higher eukaryotes, a phospho-epitope (recognized by the 3F3/2 antibody) is present on unattached kinetochores (Fig. 7).^107,113 Attachment of microtubules to a kinetochore induces dephosphorylation of the 3F3/2 phospho-epitopes at that kinetochore. As chromosomes attach to the spindle, the 3F3/2 epitopes are dephosphorylated, and the checkpoint becomes inactive . The molecular basis of this phosphorylation is not understood, but mechanistically, it is thought that checkpoint sensors, that are tension-sensitive complexes residing within the kinetochores, control the kinetochore phosphorylation status.^{105,107,112,114} Elegant studies have shown that tension exerted on kinetochores, applied by manipulating chromosomes with a micro-needle, induces loss of the kinetochore 3F3/2 epitopes.¹¹⁵ Therefore, it appears as though a lack of tension generates the checkpoint signal.

Figure 7

Tension-sensitive kinetochore phosphorylation. The kinetochores of prometaphase chromosomes (a-c and the left cell in g) become phosphorylated forming a 3F3/2 antibody-reactive epitope (open circles). The phospho-epitope is lost (filled circles) as chromosomes (more...)

The identity of the kinase which creates the 3F3/2 epitope is not known, but recent work indicates that it is an integral component of kinetochores. Cells lysed in detergent do not contain kinetochores that are reactive against the a-3F3/2 antibody, but the a-3F3/2 reactivity can be reinstated by the addition of ATP.^116,117 The activity of the kinase must be tightly associated with kinetochores. Furthermore, the substrate and kinase are likely to be associated. In theory, this “in vitro” system could be used as a biochemical assay to identify the kinase.

Genetic studies have revealed components of the yeast spindle assembly checkpoint (Fig. 8). Several groups of checkpoint proteins were identified in genetic screens designed to find mutants sensitive to microtubule antagonists. These are Mad1, Mad2, Mad3 (Mitotic Arrest Defective),¹⁰⁹ and Bub1, Bub2, Bub3 (Budding Uninhibited by Benzimidazole).¹¹⁸ In addition, Mps1 is required.¹¹⁹ Many of these proteins have homologs in higher eukaryotes (see Table 1). One of these proteins, Mad2, was shown to bind selectively to phosphorylated kinetochores in vertebrate cells.¹¹⁷ Conversely, Mad2 binding was inhibited by kinetochore-microtubule attachment.¹²⁰ Therefore, phosphorylated components of attachment-sensitive or tension-sensitive complexes might be recognized by Mad2. The current hypothesis is that Mad2 binding to 3F3/2-positive epitopes leads to the formation of an active checkpoint complex. In this model, kinetochores are catalytic sites for formation of the checkpoint signaling element, namely the activated Mad2 complex.

Figure 8

Two branches of the spindle checkpoint. Mad2 and Bub2 are both activated by the kinase Mps1 but induce arrest at different stages of mitosis. The Mad2 (spindle assembly) pathway inhibits the metaphase to anaphase transition by preventing APC^Cdc20-dependent (more...)

Table 1

Mitotic checkpoint components.

The target of the activated checkpoint complex was revealed in key experiments demonstrating that cell cycle arrest is brought about by inhibition of APC activity, which in turn prevents Pds1 ubiquitination and subsequent degradation. Yeast Mad2 was shown to bind to Cdc20, a component of the APC required for Pds1 degradation,^121,122 and this binding can inhibit ubiquitination of APC substrates.¹²¹ Overexpression of CDC20, or expression of a cdc20 mutant that cannot bind to Mad2, bypasses the spindle assembly checkpoint arrest.¹²² In the catalytic kinetochore model, unattached kinetochores might form an active site at which Mad2-Cdc20 complexes are assembled, then released, thereby excluding Cdc20 from APCs. Alternatively, active Mad2 complexes might be released from kinetochores allowing them to inhibit APC^Cdc20 in other cellular locations. The latter model is supported by measurements of Mad2 localization dynamics in living cells; Mad2 is a transient component of unattached kinetochores, having a t_1/2 of roughly 25 seconds.¹²³

But what is the nature and function of the active Mad2 complex? Studies in yeast have shown that spindle defects activate kinase Mps1, resulting in Mad1 hyperphosphorylation (perhaps directly by Mps1).^119,124 Overexpression of Mps1 alone can activate the checkpoint, and this arrest is (at least partly) dependent on Mad1, Mad2, Mad3 and Bub1, Bub2, Bub3. Mad1 phosphorylation also requires Bub1, Bub3 and Mad2.^124,125 This modified form of Mad1 is required to mediate metaphase arrest. Significantly, Mad1 has been shown to bind to Mad2, and in this complex, Mad1 is a better substrate for Mps1 kinase than is unbound Mad1. At least in Xenopus egg extracts, Mad1 is required for the association of Mad2 to kinetochores that are not attached to the mitotic spindle.¹²⁶ Together, the yeast genetic data and studies in higher eukaryotes indicate that checkpoint activation relies on the recognition of unattached kinetochores by Mad2, and the formation of an activated Mad1-Mad2 complex in which Mad1 is hyperphosphorylated. But how do the other checkpoint components fit into this scheme?

Somewhat parallel to the case of the Mad1-Mad2 complex, yeast Bub1 and Bub3 are tightly associated.¹²⁷ This is also the case in mammalian cells, and the Bub1 domain required for Bub3 binding is also needed for localization of Bub1 to kinetochores.¹²⁸ The implication is that Bub3 drives localization of Bub1 to kinetochores, as is the case for Mad1 and Mad2. A recent study of the budding yeast proteins sheds some light into how these complexes might be related.¹⁰⁸ Mad1 was shown to associate with Bub1 and Bub3 in unperturbed cell cycles, and the amount of the complex in cells was increased in response to spindle checkpoint activation. Mad2 and Mps1 are required for the formation of the Bub1-Bub3-Mad1 complex in yeast.¹⁰⁸ A Mad1 mutation that abolished Bub1-Bub3-Mad1 complex formation also led to a defective spindle checkpoint.

But how are the Mad1-Mad2 and Bub1-Bub3 complexes related? It may be the case that each complex becomes localized to kinetochores under slightly different conditions, in order to broaden the scope of defects that the checkpoint can detect. However, since deletion mutants of any one of these components results in a fully defective checkpoint, it is hard to argue that the complexes play entirely redundant roles. Instead, the different complexes might well detect different aberrations, but still all be needed for generating the active checkpoint complex that inhibits APC^Cdc20. Recent studies have allowed a working model to explain such an interconnection between the Mad1-Mad2 and the Bub1-Bub3 complexes, and how APC^Cdc20 might be inhibited.¹⁰⁸ Although Bub1-Bub3-Mad1 complexes exist in yeast, and this complex forms in a manner dependent on Mad2 and Mps1 kinase activity, Mad2 is not found in this complex.¹⁰⁸ Additionally, the Bub1-Bub3-Mad1 complex does not seem to be able to bind to Cdc20.¹⁰⁸ This might suggest that an exchange mechanism is necessary to generate the active checkpoint complex (Fig. 9). In such a model, Mad2 is displaced from Mad1-Mad2 complexes, induced by Mps1 kinase, simultaneously stimulating the formation of the Bub1-Bub3-Mad1 complex on the one hand and an active Mad2 complex on the other.^108,129 Mad1 phosphorylation, dependent on Mps1, Bub1, Bub3 and Mad2, may also be involved in this exchange. The nature of the activated Mad2 complex in not known but may include Mad3.¹²⁹

Figure 9

Model for the formation of the activated spindle assembly checkpoint complex. Association of Mad1-Mad2 and Bub1-Bub3 complexes with kinetochores is stimulated by the onset of spindle assembly and/or spindle aberrations. Kinase Mps1 induces exchange between (more...)

In support of this model, animal homologues of Mad1, Mad2, Mad3, Bub1 and Bub3 are found at the kinetochores of prophase and prometaphase (not yet bioriented) chromosomes. Following congression to the metaphase plate, these proteins seem to dissociate.¹³⁰ These localization studies suggest that formation of an active checkpoint complex within kinetochores is likely to be a conserved mechanism that activates the checkpoint pathway. But how is the checkpoint signal mobilized? How does a single, unattached kinetochore generate a signal that inhibits anaphase spindle elongation and prevents loss of sister chromatid cohesion of all the other chromosomes? One study has revealed important information that should help to resolve this question. Rieder et al. examined the timing of anaphase onset in cells that contain two functional and independent spindles.¹³¹ Such polykaryons are generated by cell fusion. When two cells at different stages of the cell cycle are fused, cell cycle progression of their nuclei soon becomes synchronized, allowing measurements of anaphase timing in independent spindles that share a common cytoplasm. This analysis revealed that the inhibitor emanating from a single unattached kinetochore is not freely diffusible, but rather is likely to be associated with the spindle itself. Therefore activated complexes might track from kinetochores along spindles.

Checkpoint Control Of Mitotic Exit

Many of the components of the mitotic exit machinery have been identified by the cell cycle phenotype of budding yeast mutants which arrest as a large dumbbells with elongated spindles (see Table 1 and 2). This phenotype is consistent with arrest at the anaphase/telophase transition. Mutants are unable to pass this arrest, and the proteins are therefore essential for exit from mitosis. These genes appear to define a GTP-dependent kinase signaling cascade, ultimately releasing a phosphatase that induces spindle disassembly, cytokinesis and mitotic exit. Control of mitotic exit therefore resides in the inhibition of this essential pathway.

Table 2

Function/localization of mitotic checkpoint components.

Exit from mitosis absolutely requires inhibition of B-type cyclin/Cdk activity. Under normal circumstances this is mediated by both inhibition of Cdk activity and by degradation of mitotic cyclins. Study of the S. cerevisiae spindle checkpoint has revealed that the ‘spindle assembly’ checkpoint is branched, inhibiting both the transition from metaphase to anaphase and mitotic exit (Fig. 8). The different functions of the two branches begs the question as to whether it is erudite to continue calling both branches by the term “spindle assembly checkpoint”. Others have begun to call the two branches the “spindle assembly” and “spindle position” checkpoints.¹³² For the purposes of this review we continue to use the term spindle assembly checkpoint for the inhibition of the metaphase-anaphase transition and the generic term “mitotic exit control” for the branch that regulates the activity of the B-type cyclin/Cdk activity. The mammalian machinery for mitotic exit control is currently being elucidated while substantial in-roads into understanding the mechanism has been achieved in S. cerevisiae. Here we describe current knowledge in the S. cerevisiae checkpoint control of mitotic exit (Fig. 10). Comparison to mammalian homologues and discussion of their possible clinical importance in tumorigenesis is left for the next section.

Figure 10

Mitotic exit (Bub2) pathway. Many components of the MEN localize to the spindle pole bodies either symmetrically or asymmetrically with preference for that destined for the daughter cell. Components of the RENT complex localize mainly to the nucleolus. (more...)

Evidence that Bub2 operates in a separate checkpoint pathway to the Bub1, 3, Mad1-3 pathway (hereafter collectively referred to as the Mad2 pathway) came from studies of double mutants treated with antitubulin drugs.^133-138 Double mutant combinations that included bub2D failed to arrest in nocodazole whilst double mutant combinations that did not include Bub2 retained a mitotic delay. However, in mad2D cells treated with nocodazole the metaphase-anaphase transition occurs with kinetics comparable to those of untreated cells while bub2D cells delay the metaphase-anaphase transition. In addition, delay of the cell cycle in ctf13 mutants (limited for a key kinetochore component) requires Bub1 and 3 and Mad 1, 2 and 3 but is independent of Bub2.¹³⁹ In cdc20 mutants, the mitotic arrest caused by maintained Pds1 levels is dependent on Bub2 but independent of the Mad2 pathway genes.¹⁴⁰ Inhibition of Dbf2 in late mitotic arrest requires Bub2 but not the Mad2 pathway proteins.¹⁴¹ Together, these studies define distinct pathways. The Mad2 pathway ultimately targets Pds1, preventing spindle elongation and loss of sister chromatid cohesion at the metaphase to anaphase transition. The Bub2 pathway inhibits mitotic cyclin/Cdk activity and thus prevents spindle disassembly and exit from mitosis. Since the Bub2 pathway also monitors spindle integrity and is triggered by microtubule depolymerizing agents, there is a common upstream element, kinase Mps1. However, in contrast to the inhibition of anaphase onset via APC^Cdc20 regulation mediated by the Mad2 pathway, the Bub2 pathways appears to primarily act by inhibition of mitotic cyclin degradation and maintenance of mitotic cyclin dependent kinase activity. This is achieved by suppression of APC^Cdh1 and Sic1 activity which promote Clb1/Clb2 degradation and inhibit mitotic cyclin-dependent kinase activity, respectively.

The mitotic exit branch of the checkpoint is essential if microtubule polymerization is perturbed during anaphase, i.e. after APC^Cdc20-dependent degradation of Pds1. Normal progression of the cell cycle ensures that Cdc20 remains active and bound to the APC until after the onset of anaphase, when Cdh1 replaces Cdc20 as the APC specificity factor targeting B-type cyclins for degradation. However, deletion of CDH1 is not sufficient to prevent mitotic exit since inactivation of mitotic cyclin-dependent kinase activity by Sic1 is sufficient to allow exit from mitosis. The redundancy of APC^Cdh1 and Sic1 activity ensures that cells may exit mitosis in the absence of checkpoint stimulation. In the presence of checkpoint stimulation, the activity of both Sic1 and APC^Cdh1 is inhibited by nucleolar sequestration of the phosphatase Cdc14. Indeed, it appears that release of Cdc14 from the nucleolus is a key event in mitotic exit. The mechanism of Cdc14-mediated exit from mitosis appears to be three-fold. First, by dephosphorylating Cdh1 the APC targets the B-type cyclins for degradation. Ordinarily, phosphorylation of Cdh1 by Cdc28 is inhibitory and thus is self protecting,¹⁴² but when Cdh1 is dephosphorylated by Cdc14, this self-protection is removed. Second, by dephosphorylating Swi5, activating the transcription of Sic1, and third by directly dephosphorylating Sic1 itself. Thus Cdc14 both inhibits the activity of cyclin/Cdk activity and induces the destruction of the cyclin components.¹⁴³

How does the “mitotic exit” checkpoint inhibit the release of Cdc14 from the nucleolus? Throughout most of the cell cycle Cdc14 is held inactive within the nucleolus in complex with Net1/Cfi1 (Fig. 11)^144,145 termed the RENT complex (regulator of nucleolar silencing and telophase). This inactive localization appears to be dependent upon Tem1,¹⁴⁵ a GTP binding protein localized to the daughter-bound (SPB). Cdc14 may also play a structural role in the nucleolus.¹⁴⁶ A recent paper proposes a mechanism for monitoring the completion of anaphase and presents a compelling model.¹⁴⁷ When SPB-associated Tem1-GDP locates to the bud at the end of anaphase, it interacts with a cortical protein Lte1, which is a GDP/GTP exchange factor (GEF). Tem1-GDP is activated by conversion to Tem1-GTP triggering the release of Cdc14 via a kinase cascade termed the mitotic exit network (MEN see below). Even when localized to the bud cortex, Tem1 activation could be inhibited by GAP (GTPase activating protein) activity of Bub2, preventing exit from mitosis. There are several compelling reasons for supposing this to be the checkpoint mechanism. Bub2 has considerable sequence homology to cdc16 in S. pombe, which is known to form a two-component GAP with byr4. Together they activate the GTPase encoded by spg1, the S. cerevisiae homolog of Tem1. Furthermore, deletion of the cerevisiae homolog of byr4 (Bfa1) causes a phenotype similar to bub2D, as does overexpression of Tem1. Finally, localization of Bub2 to the SPB and preferentially to that destined for the daughter cell,^138,148,149 provides strong circumstantial evidence in support of this model.

Figure 11

Model for colocalization of Tem1/Lte1 inducing mitotic exit in budding yeast. Mitotic exit is triggered by Tem1-GTP that induces release of Cdc14 from the nucleolus. Inactive Tem1-GDP is bound to the SPB destined for the daughter cell. Once the spindle (more...)

The mechanism by which activation of Tem1 leads to the release of Cdc14 from the nucleolus via the MEN involves a number of key components including Cdc15, Cdc5, Dbf2, Dbf20 and Mob1.¹⁵⁰ Most appear essential for mitotic exit since single mutants are lethal, while in the case of Dbf2 and Dbf20, it is only the double mutant that is synthetically lethal.¹⁵¹ Localization and phosphorylation of these proteins appear to be important factors during the cell cycle and probably contribute to their function (Table 2). In particular, many of the components localize to the nucleolus or are asymmetrically distributed between the SPBs, being preferentially bound to that destined for the daughter cell. Cdc15 localizes to the SPB during mitosis and relocates to the bud neck after telophase.^152,153 Furthermore, Cdc15 phosphorylation increases gradually during the cell cycle until it is rapidly dephosphorylated in late mitosis.^152,154 Like Cdc15, Dbf2 localizes to the SPB and moves to the bud neck in telophase.¹⁵⁵ While asymmetric localization to the daughter-bound SPB is true for some components, others such as Mob1 (or at least the S. pombe homolog), localize symmetrically to both spindle pole bodies. Localization to the SPB and the relocation of these components to the bud neck in telophase mimics the localization in S. pombe in which their homologs principally act by regulating cytokinesis rather than mitotic exit per se.

While mechanistic details of the MEN/RENT complex are being reported, much work has still to be completed before a clear picture can be drawn. Some details of physical interactions and co-localization can at least allow some description of the cascade (Fig. 10). The rapid dephosphorylation of Cdc15 in late mitosis appears to be mediated by Cdc14.¹⁵³ However, there also appears to be a role for Cdc15 as an activator of Cdc14, and it is thus both an activator and substrate.¹⁵⁴ Also there appears to be a role for Pds1 as an inhibitor of B-type cyclin degradation independent of its role as a securin¹⁵⁶ thus forming potential “crosstalk” between the Mad2 and Bub2 checkpoint branches. What is less clear is the role of Cdc5 which physically interacts with Dbf4 (part of the DNA replication machinery) but as yet has no clearly defined role in the mitotic exit pathway. As Cdc5 is a target of DNA damage checkpoint control, this component offers an attractive link between the damage checkpoint and mitotic exit control. Direct interactions have been established for a number of MEN components while the exact nature of many interactions remain unclear. For example, physical interaction between Mps1 and Mob1 has been demonstrated. Furthermore, interactions between Mob1 and Dbf2 and Dbf20 have been demonstrated. However, there has been no direct link established between these components and the remaining MEN components to date. Similarly, the role of Cdc5 in the MEN has yet to be elucidated. Nonetheless, we present an attempt to order the events of mitotic exit regulation based upon physical interactions and localization in Fig. 10. In this scheme we present Cdc15 “upstream” of Cdc14 as the former localizes to the SPB coincident with other upstream elements.

Evidence suggests that Bub2, and its associated partner Bfa1, participate in an essential checkpoint that is also activated by DNA damage.^157,158 Thus the maintenance of B-type cyclin/Cdk activity by the Bub2 pathway may represent a universal mechanism that can respond to stress at any stage of G2 and M-phase. Other genetic interactions of Bub2 include synthetic lethality of a bub2D arc35-1 mutant¹⁵⁹ and that Bub2 is essential for arrest in tub4-1 cells.¹⁶⁰ Thus, the Bub2 pathway seems to respond to defects in spindle orientation, spindle localization, spindle damage and DNA damage.

Oncological Implications of Mitotic Checkpoint Homologs

The existence of numerous mitotic exit and spindle assembly checkpoint protein homologs in S. pombe and higher eukaryotes suggests that similar mechanisms regulate mitotic exit in all eukaryotes despite the fact that the asymmetric cytokinesis in S. cerevisiae appears to have fundamentally different spatial and temporal strategies. As aberrant mitosis frequently results in asymmetric distribution of the genetic material and aneuploid daughter cells, dysfunctional regulation of these checkpoints has become an attractive hypothetical mechanism for chromosomal instability in mammalian tumorigenesis. Indeed, some established tumor cell lines and tumors appear to have dysfunctional checkpoint controls^23,161,162 and some of the checkpoint proteins appear to be targets of oncogenic viral proteins.¹⁶³ However, screening of aneuploid colorectal tumor panels for such mutations revealed only mutations in the human hBUB1/hBUBR1 genes.¹⁶⁴ A similar study of 31 aneuploid lung, and head and neck, tumors showed no such mutations.¹⁶⁵ One hBUB1 somatic mutation that led to an amino acid substitution was found among 30 human primary lung cancer tumors.¹⁶⁶ hBUB1 and hBUBR1 mutants have also been found in adult T-cell leukemias/lymphomas¹⁶⁷ and some colorectal tumor cell lines.¹⁶¹ Perhaps significantly, one study has implicated Brca2, which is responsible for a fraction of the inherited susceptibilities to breast cancer, in the spindle assembly checkpoint.¹⁶⁸ Brca2 was found to interact with hBubR1 and was phosphorylated by hBubR1 in vitro, though no direct role in the checkpoint was demonstrated. Although inactivation of Bub1 appears to confer chromosomal instability,¹⁶¹ more studies are required to determine whether mutations in BUB1 and other mitotic checkpoint proteins represent significant causative events or whether other checkpoints may account for aneuploid tumorigenesis.

In addition to the MAD2 and BUB2 pathway components, a number of other S. cerevisiae genes appear to have mammalian homologs which have been implicated as either proto-oncogenes or as tumor suppressor genes (see Table 1). Notably S. cerevisiae Pds1 may have two mammalian homologs, at least one of which is associated with pituitary tumors.^169-172 Most of the human genes have been localized at least to the chromosome level, and recent publication of the human genome will therefore facilitate further study. Furthermore, many mouse homologs have been identified, and murine models of tumorigenesis may further elucidate the contribution of these genes to tumorigenesis. At the present time, only two knockout mouse models have been reported, MAD2¹⁷³ and BUB3,¹⁷⁴ both of which are early embryonic lethals. In the BUB3 mouse, from day 3.5 onwards, embryonic cells display mitotic aberrations such as micronuclei, anaphase chromosome laggards and bridging. In the presence of microtubule antagonists, the cells fail to arrest in metaphase.

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