There are two classes of yeast mutants in which cells enter mitosis ... in DNA repair [17,18]. Navas et al. ... only those mutations that affect the polymerase E car-.
CELL-CYCLE CHECKPOINTS CELL-CYCLE CHECKPOINTS
HUMPHREY AND TIM HUMPHREY Tim AND TAMAR TAMAR ENOCH ENOCH
Keeping mitosis in check Mutations in an essential yeast gene, encoding DNA polymerase E, abolish the dependence of mitosis on the completion of DNA replication, suggesting that the replication complex provides the checkpoint signal. The events of the eukaryotic cell cycle - such as DNA replication, chromosome condensation and cell division - can each be arrested if earlier cell-cycle events have not been completed, or if damaged DNA is present. Hartwell and Weinert [1] proposed that arrest is achieved through controls called "checkpoints", which can monitor cellular events and delay cell-cycle progression if necessary. By definition, mutants deficient in these checkpoints progress through the cell cycle under conditions that would normally induce arrest. Efficient checkpoint control depends on a multi-step process, analogous to signal transduction, that allows information to be 'transmitted' between the functionally distinct protein complexes responsible for controlling different aspects of the cell cycle. One approach to the study of checkpoints has been the identification of checkpoint-deficient mutants in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (reviewed in [2]). In this article, we focus on the yeast checkpoint that maintains the dependence of mitosis on the prior completion of DNA replication.
(see Table 1). The first class includes mutations in certain S. pombe rad and hus genes, and S. cerevisiae MEC, RAD and SAD genes (Table 1) [3-7]. The checkpoint defect in these mutants is revealed by treating cells with inhibitors of DNA replication. Instead of undergoing cell-cycle arrest, the mutant cells proceed into mitosis and attempt to segregate a single set of chromosomes, with lethal consequences. These mutants are otherwise viable, although in some cases complete deletion of the relevant gene is lethal [5]. As these class 1 checkpoint mutants are viable, they are unlikely to be have defects in replication or mitosis. Instead, the mutations must disrupt the pathway that ensures the interdependence of these two processes (see Fig. 1). Many of these mutations also abolish the ability of cells to arrest in response to damaged DNA, suggesting that replication and damage are monitored by similar processes. Although many of the genes affected by these mutations have been cloned and sequenced, so far this has provided little information on the molecular mechanisms underlying checkpoint control.
There are two classes of yeast mutants in which cells enter mitosis without first completing DNA replication
The second class of mutations disrupts functions that are essential for the initiation of DNA replication in S phase
Fig. 1. Model for the molecular basis of the phenotypes of different classes of checkpoint mutant. In wild-type cells, the presence of a replication complex activates a checkpoint that inhibits mitosis. Class 1 mutants have defects in this checkpoint pathway. Class 2 mutants fail to assemble a replication complex, so the checkpoint is not activated. Polymerase E mutants, which are viable but checkpoint-defective, have lost the ability of the replication complex to activate the checkpoint. 376
© Current Biology 1995, Vol 5 No 4
DISPATCH
(see Table 1). Included in the second class are mutations in the S. pombe cdcl8, cut5/rad4 and cdtl genes [8-11]. These mutants are not viable, as they are unable to synthesize DNA. However, instead of undergoing cell-cycle arrest, like many other S-phase-defective yeast mutants, the cells with class 1 mutations progress into mitosis. Thus, in addition to disrupting DNA replication, these mutations also abolish the checkpoint. Recently, deletions of the S. cerevisiae CDC6 gene have been shown to have a similar phenotype (Piatti and Nasmyth, personal communication). To explain this phenotype, it has been proposed that assembled replication complexes activate the checkpoint pathway, thus inhibiting mitosis while replication is in progress (Fig. 1). In mutants of class 2, replication complexes are not assembled and so no inhibitory signal is generated to arrest the cell cycle [8-12]. This model predicts that some component of the replication complex interacts directly with the checkpoint 'signal transducing' machinery. If this is the case, it should be possible to isolate mutants in replication factors that are specifically unable to transduce a checkpoint signal, but are able to perform DNA replication normally. A mutation in the
S. cerevisiae gene encoding DNA polymerase e, recently reported by Navas et al. [13], has these properties. The identification of this mutant establishes that the replication complex is directly involved in checkpoint control (see Fig. 1). The polymerase E mutant was isolated in a screen for S. cerevisiae mutants defective in the transcriptional response to agents that damage DNA or block replication. In response to such treatments, transcription of the RNR3 gene (which encodes a subunit of ribonucleotide reductase, RNR) is induced in wild-type cells. Mutants which failed to induce RNR transcription were identified; these dun (damage uninducible) mutants fell into five complementation groups. Cells with mutations in one gene, DUN2, also failed to arrest mitosis when S-phase was blocked. The dun2 mutations render cells temperature-sensitive for growth, although the checkpoint and transcriptional defects are observed at the permissive temperature. Sequence analysis and classical genetics revealed that DUN2 is identical to POL2, which encodes the catalytic subunit of DNA polymerase E, one of three polymerases
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Current Biology 1995, Vol 5 No 4 essential for DNA replication in S. cerevisiae [14-16]. Other studies suggest that polymerase can perform leading-strand DNA synthesis and may also be involved in DNA repair [17,18]. Navas et al. [13] examined other available mutations in the POL2 gene and found that only those mutations that affect the polymerase E carboxyl terminus abolished the checkpoint that monitors unreplicated DNA. All these mutations are nonsense mutations, resulting in truncations of polymerase E by removing sequences immediately carboxy-terminal to a zinc-finger domain that is conserved between the yeast and human proteins [19]. In contrast, strains with mutations affecting the polymerase's amino terminus display defects in DNA replication at the restrictive temperature, but arrest normally when DNA replication is blocked at the permissive temperature. The carboxyl terminus contains a zinc-finger motif which is unique to polymerase e, and is thus unlikely to be involved in the polymerization of nucleotides that all polymerases perform. Furthermore, the checkpoint mutations do not abolish DNA replication. It is therefore reasonable to propose that the carboxyl terminus of polymerase E triggers a biochemical pathway that culminates in mitotic arrest. Moreover, it is also likely to trigger a distinct pathway that culminates in transcriptional activation of damageinducible genes. Thus, this region of polymerase E seems to play a central role in coordinating the response of cells to failure to complete S phase. Phenotypically, dun2 mutations in polymerase E resemble mutations in the rad, MEC and hus genes classl genes; see Table 1) which disrupt the checkpoint without affecting S phase or mitosis. Perhaps these mutations disrupt a checkpoint that is activated only when S phase is arrested artificially. Alternatively, the checkpoint could function during every cell cycle, blocking mitosis until DNA replication is complete. Strains lacking polymerase E are known to be nonviable and deficient in DNA replication [14]. If the checkpoint is only required when S phase is arrested artificially, complete deletion of the polymerase E gene could result in normal cell-cycle arrest. This would imply that the polymerase -dependent checkpoint is required only for response to unusual stimuli. However, if cells bearing the polymerase E deletion enter mitosis (like cells with mutations in the cdc18, cut5/rad4 and cdtl genes), it would seem that polymerase E might also activate the mitotic checkpoint during normal cell cycles. What role is the carboxyl terminus of polymerase playing in the checkpoint pathway? Navas et al. [13] propose that it works as a sensor, detecting regions of singlestranded DNA that form when DNA polymerase progression is blocked. The dun2 mutation, which affects a site close to the zinc-finger DNA-binding motif, may abolish the ability of this motif to bind and 'sense' singlestranded DNA. Alternatively, the carboxyl terminus of the polymerase could participate in protein-protein interactions that activate the checkpoint pathway. These interactions might only take place when polymerase is
assembled in a replication complex. According to this model, the carboxyl terminus functions more as a signal than as a sensor. To distinguish between these models, genetic, biochemical and molecular techniques could be used to identify factors that interact with the carboxyl terminus of polymerase ; the role of any such proteins in the checkpoint could then be assessed. It will also be important to determine whether any of the MEC, rad or hus gene products interact with the polymerase E carboxyl terminus. Disruption of checkpoint controls in the cells of higher eukaryotes has been proposed to promote tumorigenesis by increasing genetic instability [20]. Mutations in the tumor suppressor gene p53 have been shown to disrupt the checkpoint that ensures cell-cycle arrest at the G1- to S-phase boundary in response to DNA damage, and also to promote gene amplification and tumorigenesis (reviewed in [21]). The carboxyl terminus of polymerase E has been conserved between S. cerevisiae and humans [19], suggesting that its role in checkpoint control is also conserved. Could carboxy-terminal truncations of the human DNA polymerase E increase genetic instability and thus promote tumorigenesis? Perhaps studies in yeast are pointing yet again to a novel class of tumor suppressors. References 1. Hartwell L, Weinert T: Checkpoints: controls that ensure the order of cell cycle events. Science 1989, 246:629-634. 2. Murray AW: Creative blocks: cell-cycle checkpoints and feedback controls. Nature 1992, 359:599-604. 3. Al-Khodairy F, Carr AM: DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO 1992, 11:1343-1350. 4. Al-Khodairy F, Fotou E, Sheldrick KS, Grifiths DJF, Lehmann AR, Carr AM: Identification and characterisation of new elements involved in checkpoint and feedback controls in fission yeast. Mol Biol Cell 1994, 5:147-160. 5. Allen JB, Zhou Z, Siede W, Friedberg EC, Elledge SJ: The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev 1994, 8:2416-2428. 6. Enoch T, Carr AM, Nurse P: Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev 1992, 6: 2035-2046. 7. Weinert TA, Kiser GL, Hartwell LH: Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev 1994, 8:652-665. 8. Kelly TJ,Martin GS, Forsburg SL, Stephen RJ,Russo A, Nurse P: The + fission yeast cdc18 gene product couples S-phase to START and mitosis. Cell 1993, 74:371-382. 9. Hofmann JFX, Beach D: cdtl is an essential target of the Cdc10/Sct1 transcription factor: requirement for DNA replication and inhibition of mitosis. EMBO J 1994, 13:425-434. 10. Saka Y, Yanagida M: Fission yeast cut5+ , required for S-phase onset and M-phase restraint, is identical to the radiation-damage repair gene rad4+ . Cell 1993, 74:383-393. 11. Saka Y, Fantes P, Sutani T, Mclnerny C, Creanor J, Yanagida M: Fission yeast cut5+ links nuclear chromatin M phase regulator in the replication checkpoint control. EMBOJ 1994, 13:5319-5329. 12. Li JJ, Deshaies R: Exercising self-restraint: discouraging illicit acts of S and M in eukaryotes. Cell 1993, 74:223-226. 13. Navas TA Zheng Z, Elledge SJ:DNA polymerase e links the DNA replication machinery to the S-phase checkpoint. Cell 1995 74: 29-39. 14. Morrison AL, Araki H, Clark AB, Hamatake RK, Sugino A: A third essential polymerase in S. cerevisiae. Cell 1990, 62:1142-1151. 15. Araki H, Ropp PA, Johnson AL, Morrison A, Sugino A: DNA polymerase II, the probable homolog of mammalian DNA polymerase e, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBOJ 1992, 11:733-740.
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Saccharomyces cerevisiae. Nucleic Acids Res 1994, 22:3104-3112. 23. Enoch T, Nurse P: Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell 1990, 60:665-673. 24. Sheldrick KS, Carr AM: Feedback controls and G2 checkpoints: Fission yeast as a model system. BioEssays 1993, 15:775-782. 25. Rowley RS, Young SP: Checkpoint controls in Schizosaccharomyces pombe: radl. EMBO 1J1992, 11:1335-1342. + 26. Jimenez G, Yucel J, Rowley R, Subramani S: The rad3 gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair. Proc Natl Acad Sci USA 1992, 89:4952-4956.
Tim Humphrey and Tamar Enoch, Department of Genetics, Harvard Medical School, Warren Alpert Building, 200 Longwood Ave; Boston Massachusetts 02115, USA.
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