Annu. Rev. Cell Bioi. 1991. 7:227-56 Copyright © 1991 by Annual Reviews Inc. All rights reserved

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CELL CYCLE REGULATION IN THE YEASTS SACCHAROMYCES Annu. Rev. Cell. Biol. 1991.7:227-256. Downloaded from www.annualreviews.org by George Mason University on 03/08/13. For personal use only.

CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE Susan L. Forsburg and Paul Nurse ICRF Cell Cycle Group, M icrobiology Unit, Biochemistry Department, Oxford University, Oxford OXI 3QU, England KEY

WORDS:

cyclin, cdc, mitosis, DNA replication

CONTENTS INTRODUCTION............................. .................................................................................

227

.................................................................................................

228 228 231 232

THE yEASTS..................

Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... ........ . . . . . ....... . . . . . . . . . . . .... . . . . . . . . . . ........ Schizosaccharomyces pombe ...................................................................................

Too l s o f Ge ne tic s ......................................................................................................

REGULATION OF CELL CYCLE PROGRESSION

..•••................................................................

GljS Tr a n sitio n........................................................................................................ G2jM Tra nsitio n....................................................................................................... Exit fro m the Cyc le ..................... ............................................................................. CONCLUSIONS............. ............... ............. .......................... .. ...........................................

235 236

243 249

250

INTRODUCTION

The cell cycle is the complete process of DNA replication, mitosis, and cytokinesis that leads to the production of two daughter cells from a single mother cell. This cycle is typically divided into four phases. The events of DNA replication (S phase) and mitosis (M phase) are separated by gaps of varying length called Gl and G2. All cell types undergo some version of this basic cycle, although details of regulation and the size of the gap phases differ. Our understanding of how the progression through the cell 227 0743-4634/91 /1 1 1 5-0227$02.00

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FORSBURG & NURSE

cycle is regulated has advanced quickly in the last several years, as data from genetic systems (primarily the fungi) and biochemical systems (pri­ marily the eggs of Xenopus and marine invertebrates) have converged to form a cohesive model. Given this profusion of new information, it is impossible to exhaustively review the cell cycle field in this chapter. The reader is directed to the review by McIntosh & Hering in this volume, as well as recent reviews by Nurse (1990), Cross et al ( 1 989), Laskey et al ( 1989), and Murray & Kirschner ( 1 989). Our purpose here is to review a specific part of this field: what has been elucidated about the control of cell cycle progression in the genetically tractable yeasts. In doing so we hope to convey the usefulness of applying a genetic experimental system to cell biology questions. One of our principal concerns is the meaning of the term "regulatory function." By this we mean a gene function that specifically controls progression through the decision points of the cycle: a rate-limiting determinant. These decision points occur at the transitions between the G I and S phases and between the G2 and M phases. However, a function required for progression through the cycle does not necessarily define a regulatory checkpoint, and we will examine this concept more rigorously as we proceed. First, however, we define the distinct biologies of the two evolutionarily diverse yeasts: the budding yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. THE YEASTS

The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are simple, single-celled fungal eukaryotes with a DNA content only four to five times greater than that of Escherichia coli. Despite their simplicity they have most of the basic features typical of more complex eukaryotes. The two yeasts are as evolutionarily divergent from one another as either is from mammalian cells (Sipiczki 1 989). This divergence is reflected by differences in their life-styles and cell cycle regulation. As we will discuss, S. cerevisiae modulates its cycle chiefly at G 1 IS, whereas S. pombe regulates its cell cycle progression chiefly at G2/M . Genetic studies have nevertheless revealed that each yeast contains both control points, even though one is usually cryptic in rapidly growing cells. A compare-and-contrast approach will be used in the rest of the review as we progress through a single cycle of cell division. However, we begin by introducing each yeast separately. Saccharomyces cerevisiae

The major events of the cell cycle of S. cerevisiae are diagrammed in Figure lA (for reviews, see Byers 198 1 ; Pringle & Hartwell 198 1). The yeast grows

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cerevisiae (A) and S. pombe (B). At the bottom is a cartoon of a haploid cell, and its position relative cerevisiae, the mitotic spindle phases overlap, while in S. pombe, these phases are distinct. The S. pombe diploid is unlikely to undergo a mitotic

A schematic of the life cycles of S.

to the stages of the cell cycle. The stages are essentially similar in both haploid and diploid cells. Note that in S. forms very early and Sand M

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230

FORSBURG & NURSE

as a stable haploid or diploid cell and produces a small bud which, after initiation, grows continuously through the cycle although it does not quite reach the size of the mother cell. The nucleus migrates into the bud neck and there undergoes mitosis although the nuclear envelope remains intact, and there is no apparent chromosome condensation. This distinctive cellu­ lar architecture places some constraints upon the S. cerevisiae cell cycle. The duplication of the spindle pole bodies and the reorganization of interphase cytoplasmic microtubules into a mitotic nuclear spindle must take place early in S. cerevisiae to allow bud formation and migration of the nucleus to the bud neck. For this reason, S. cerevisiae undergoes much of the cycle with what in other systems would be cytological markers of late G2/M. Thus the budding yeast lacks clear definition between S, G2, and M phases. These phases are shown as overlapping in Figure lAo A rigorous definition of when S. cerevisiae starts M has not been agreed upon and, indeed, is an issue of contention (Nurse 1 985). The cdc mutations isolated first by Hartwell and his colleagues (Hartwell 1 974, 1 978; Pringle 1 978) identified gene functions necessary for transit through specific points in the cell cycle. That is, cells deficient in the function of a CDC (cell division cycle) gene characteristically arrest at a distinct and identifiable point in the cell cycle. These blocks mark the point beyond which the cell is unable to progress further in the cycle without the missing function. Mutations in genes necessary for progression have been identified throughout the entire S. cerevisiae cell cycle. S. cerevisiae chiefly regulates its cycle at G 1 . The regulatory network coordinates the cycle of division with environmental constraints such as nutrition and cell size and monitors these parameters chiefly in G I ; it is this phase that cells generally extend when nutritionally deprived (Pringle & Hartwell 1 98 1 ). The regulatory network also coordinates the different aspects of division with one another. Bud growth initiates at the same time as DNA replication and mitotic spindle formation, and since these may be separated from one another genetically, they may be considered as three parallel pathways coordinated in the division cycle (Pringle & Hartwell 198 1). One important feature of cell cycle regulation is that the cells must maintain the ability to leave the cycle and undergo alternative fates when conditions for continued division are not promising. In budding yeast, these alternatives are quite simple. During each G 1 , the yeast cell has three choices. First, it can proceed through the cell cycle and divide. Second, if nutritionally deprived, it can enter stationary phase where it becomes resistant to heat and chemical treatment. When such a cell is returned to a rich medium, there is a lag phase before it can re-enter the cycle (Pringle & Hartwell 1 98 1) . This dormant state allows the cells to await a return to

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CELL CYCLE REGULATION IN YEASTS

23 1

better conditions. Third, if the cells are haploid, they can mate. During conjugation, two cells of opposite mating type (a and ()() synchronize their cell cycles with a transient arrest at G 1 in response to peptide mating pheromones. Two haploids will fuse to form a stable diploid, which will continue to divide in its own mitotic cycle. If a diploid cell is starved of nutrients, however, it has the option of undergoing a meiotic cycle to pro­ duce four haploid spores. S. cerevisiae mates readily, and wild-type hap­ loids are capable of switching their mating type in order to maximize the chance of diploid formation (review, Thorner 1 98 1 ; Herkowitz & Oshima 1 98 1). The process of meiosis and sporulation forms a distinct form of cell division cycle that has similarities and differences with mitotic progression. Schizosaccharomyces pombe

The fission yeast S. pombe is a eukaryotic microbe that is, like the budding yeast, convenient for genetic and molecular analysis. Its cell cycle is more similar to that of higher cells, however, in that there are distinct G l and G2 phases (Figure IB; review, Nurse 1 985; Fantes 1 989). S. pombe grows as a rod-shaped cell of constant diameter and divides by septation and medial fission. As in other eukaryotes, microtubular reorganization and formation of the mitotic spindle take place in G2 (Hagan & Hyams 1988). The three chromosomes also condense for mitosis, although as in S. cerevisiae, the nuclear envelope remains intact throughout (review, Robi­ now & Hyams 1 989). In rapidly growing S. pombe cells, S phase begins before cytokinesis is complete, and there is only a very short G 1 phase. This reflects the fact that under these conditions S. pombe exerts cell cycle control chiefly at the G2/M transition, where information about cell size and nutrition is monitored (Fantes 1 977; Miyata et al 1 978; Fantes & Nurse 1 977). If the cell size is unusually small because of mutation or nutritional deprivation, then the fission yeast extends its G 1 phase to allow the cells to reach a critical size before they cross the G 1 IS transition (Nurse 1 975; Nurse & Thuriaux 1 977; Nasmyth 1 979; Nasmyth et aI 1979). Mutations arresting progression through the cycle, analogous to those in the budding yeast, have been isolated and the corresponding genes are likewise called cell division cycle (cdc) genes (Nurse 1 975; Nurse et a1 1 976; Nasmyth & Nurse 1 98 1). S. pombe cells continue to grow mitotically until nutrients become depleted. At such a time, cells can undergo either of two alternative fates. They may arrest in G 1 or G2, depending on temperature and the limiting nutrient, and enter stationary phase (Costello et al 1 986; review, Fantes 1 989). If cells of both mating types (h+ and h-) are present, however, or if the cells are capable of mating type switching (Klar 1 990; review, Egel 1 989), the fission yeast will undergo conjugation to form a diploid, which

232

FORSBURG & NURSE

immediately sporulates. Conjugation requires diffusible mating phero­ mones, as in the budding yeast, but also requires the cells to be nutritionally deprived. Therefore, in S. pombe conjugation, meiosis, and sporulation combine in an emergency response to nutrient limitation. The S. pombe diploid is very unstable, but it can be maintained by returning the new diploid cells to rich media before they have a chance to begin meiosis, or by appropriate mutations at the mating type locus.

Tools of Genetics There are several reasons why yeast genetics provides a powerful tool for analyzing phenomena of cell biology such as the control of the cell cycle. Genetic analysis usually begins with the isolation ofmutants, which are altered in behavior concerning the phenom­ enon of interest; these mutants may be isolated even though nothing is known concerning the biochemical basis of the phenomenon. Because the cells of both yeasts are stable as haploids, recessive mutations can be isolated and the phenotype easily examined. Essential genes can be defined by conditional mutations; in the most common, the mutant gene phenotype is revealed by a shift to high temperature (temperature-sensitive or ts mutants). The generation time of either yeast is a few hours in most conditions, and large numbers of cells can be screened using standard microbial genetic techniques. Linkage between genes may also be deter­ mined by examination of meiotic products. Subsequent molecular analysis is possible because the mutations identify the genes of interest, which can then be physically isolated by using gene banks and selecting for clones that functionally complement the mutations. Cloning is facilitated by vector systems that allow a plasmid to replicate as a stable, often high copy episome, and transformation is easy and of high efficiency (Sherman et al 1 986; Moreno et al 1 99 1). The proteins encoded by these genes can be predicted by DNA sequencing and by raising antibodies against the gene product expressed in E. coli, or another expression system. This procedure provides a route from study of the phenomenon in the intact cell (an initially rather abstract concept) to a biochemical explanation (an increasingly concrete description). Such procedures can also be used to ask very precise questions con­ cerning the causal relations between a particular molecule and its effects on the cell, by deletion, or site specific mutation of the encoding gene. This approach is analogous to the use of biochemical inhibitors or activators, either with in vitro or intact cell systems, but has the advantage of speci­ ficity. For example, inhibitors of protein kinases and phosphatases have been important in establishing that protein phosphorylation is involved in regulating cell cycle progression, but it is very difficult to interpret their

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THE POWER OF GENETICS

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CELL CYCLE REGULATION IN YEASTS

233

effects if multiple kinases and phosphatases are involved. However, molec­ ular genetics allows the modification of one particular protein kinase or phosphatase, and the specific effects of these modifications on the behavior of the intact cell can be assessed. This approach can be applied to the function of any protein once its encoding gene has been cloned. Moreover, homologous recombination occurs at a high frequency in S. cerevisiae and at a moderate frequency in S. pombe, which allows precise transplacement of modified genes back into the genome. Analyzing the functions of genes from heterologous systems is also enabled by these techniques, which thus prove to be very powerful. Genetics also allows the identification of further molecules that interact directly or are within the same pathway as the initial gene product of interest. This is possible by isolation of extragenic suppressors (review, Hotstein & Maurer 1 982). A new gene may suppress a mutant phenotype of another gene when it is also mutated, or when it is present in high copy. Suppression of a loss-of-function phenotype may identify a bypass mechanism to the function of interest, or components of a regulatory network acting upstream of the original mutant, or a component of an interacting protein complex. Dominant negative mutations, which have a damaging alteration or gain of function (Herskowitz 1 987), are also candidates for suppression. Protein interactions predicted by the sup­ pressor analysis can be confirmed with subsequent biochemical analysis by conventional protein purification, affinity chromatography co-immuno­ precipitation, and the like. CDC MUTANTS The cell division cycle mutants isolated in the two yeasts have allowed the identification of a variety of functions necessary for cell cycle progression. A cdc mutation arrests the cell at a specific point in the cell cycle. These mutations block progression through the cycle without blocking general cell growth and macromolecular synthesis, and they were originally isolated by visual screens that exploited the ability of the cells to continue growing when arrested. In S. cerevisiae, this means, for example, that cells arrest with a uniform bud phenotype. In S. pombe, this increase in mass is quite obvious; cdc mutants are highly elongated relative to the normal rod-shaped cell. In addition, mutants blocking in M phase have been identified using visual screens for specific mitotic defects (e.g. Hirano et al 1 986; Yanagida et al 1 986). Mutations in biosynthetic genes can also block progression through the cell cycle, but only as an indirect consequence of blocking growth and macromolecular synthesis. By their very nature, cell division cycle genes are essential genes and the mutations isolated in the cdc genes are conditional mutations. This leads to an important caveat: the conditional mutation by definition cannot be

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a complete loss of function, or null mutation, for it must be functional under permissive conditions. Even under restrictive conditions, the pheno­ type may not be the same as that of a null, for the gene function may be changed rather than destroyed, or some partial function may leak through. The conditional phenotype allows easy selection for cloning. This selection also allows use of suppressor analysis (described in the previous section). Several cell cycle genes have been isolated by their ability to suppress the mutant phenotype of another gene, either when the new gene is also mutant, or when it is overproduced on a high copy vector. Thus a mutant phenotype in one of the yeasts acts as a starting point from which many additional and interacting gene products may be identified and from which the formal relationships between the gene products may be established. Another tool necessary to analyze the cycle is the ability to synchronize a population of cells in culture at a particular point in the cell cycle by selective or inducing methods. This is useful for determining fluctuations throughout the cycle in protein and RNA synthesis, protein modification, and associations between proteins. This synchrony may be selected by isolating cells on the basis of size, for example by centrifugal elutriation. Synchrony can also be induced. For example, shifting tem­ perature-sensitive cells to a restrictive temperature for certain cdc mutants will block them all at the cell cycle arrest point of that mutant. After a few hours at the restrictive temperature to ensure that all cells have reached the block point, the cells can be shifted back to the permissive temperature at which they will recover and return to the cell cycle from the same point. Chemical treatments can also induce synchrony. Hydroxyurea blocks cells in S phase by blocking DNA replication. In S. cerevisiae, the purified peptide mating pheromone alpha factor can be used to synchronize cells at G l and then washed free to return cells to the cycle. There are problems, however, particularly in inducing a cell cycle block and then releasing it, as the physiological perturbation on the system cannot be fully determined, and an effect on the phenomenon of interest cannot be ruled out. Moreover, the synchrony only lasts for a few generations before the cells drift apart. By making use either of conditional mutants with different restrictive conditions, or of other means of inducing cell cycle arrest, gene functions may be ordered relative to one another or to exogenous block points (such as alpha-factor or hydroxyurea-induced arrests). This is not always straightforward because it requires that the conditions used to inactivate one gene do not affect the activity of the others and that under the arrest conditions a pathological condition does not develop that can influence the ability to recover. SYNCHRONY

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CELL CYCLE REGULATION IN YEA"STS

235

NOMENCLATURE At this point we make a note about terminology. The gene names of the two yeast systems are different. In S. cerevisiae, genes are indicated by three italicized capital letters and a number when wild type (e.g. CDC28) and by lowercase letters when mutant (cdc28). There is an exception when the mutant allele is dominant, in which case it also occurs in capital letters, but with an allele designation (e.g. DAFl-I). In S. pombe, all genes are designated by three lowercase italic letters and a number, and wild-type genes have an appended plus (e.g. cdc2+ ). Protein products are indicated by non-italicized gene names. Much confusion is engendered by the fact that the same cdc number in fission and budding yeast designates a completely different gene. By describing the different nomenclature systems, we hope it will be clear to which we refer. To assist in our endeavor, Table I describes our regulatory dramatis personae and gives a list of homologues between the two systems.

REGULATION OF CELL CYCLE PROGRESSION

How do we define a regulatory gene? The term can be misleading. Not every gene that gives a cell cycle arrest phenotype when mutated is a regulatory gene; phrased another way, not every gene whose function is necessary for cell cycle progression defines a regulator. For example, the gene encoding DNA ligase is a cdc gene which, if mutated, blocks cell cycle progression during S phase. But DNA ligase is unlikely to be a regulator. Thus a regulatory gene is not defined necessarily by a block in progression, but rather because its mutation confers inappropriate pro­ gression. Such inappropriate regulation could be characterized as an aberrant response to some signal: a failure to respond appropriately to nutrient limitation, or relief of the dependency of M phase upon the completion of S are both examples of inappropriate regulation. The pur­ pose of a regulatory checkpoint is to prevent cycle progression unless certain conditions are met. The hallmark of a regulatory mutant is to proceed despite a signal to halt. A search for such altered regulation can be expected to reveal the rate-limiting steps that actually control progression rather than merely enable it. In the rest of this review we go through a complete cell cycle, from START to finish. We evaluate the regulators that act at each phase, and characterize their interface with the environment, with other signals in the cell, and with each other. We only consider cells in a growing state that have already made the transition from stationary phase to G 1. Regulation in such cells occurs at the critical transition points between the adjustable

236

FORSBURG & NURSE

Table 1

Major cell cycle regulatory genes and homologues in S. cerevisiae and S. pombea

S. cerevisiae gene

S. pombe gene

Function (stage of cycle)

...

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CDC28

cdc2+

p34 protein kinase G I/S and G2/M

CKSl

sucl+

Function unknown CKSI: G I /S (?) sucJ+: G2/M (?) Mitotic cyclin G2/M

cdc13+ CLNI

Gl cyclins GI/S

Sequence and functional homologues; also identified in other systems Sequence homologues; also identified in human cells Sequence homologue to cyclins (B- type)

Sequence homologues to

cyclins (distinct class)

CLN2 WHIl (DAFI; CLN3) cdcJO+

Function unknown G I/S

cdc25 +

Inducer of mitosis G2/M

weel+

CDC36 CDC39

Inhibits mitosis; protein kinase G2/M Induces mitosis; protein kinase G2/M Function unknown G I /S (haploids only)

CDC37

Function unknown G I/S

MIHI

Homologiesb (function or sequence)

niml+ (cdr j+ 1 )

Partial sequence homology with notch, glpl, lin12, CDC6, SW14, SWl6 Sequence and functional homologues; also identified in other systems

, References are in the text. b A sequence homologue shares substantial similarity; a functional homologue acts appropriately in a heterologous system. References are in the text.

gaps and the working parts of the cycle, S and M . Thus the G I /S and G2/M transitions are our principal focus.

G 1 / S Transition Hartwell and colleagues defined the concept of START in budding yeast; by analogy the concept is also applied to fission yeast (Hartwell 1 974; Pringle & Hartwell 1 98 1 ; Nurse 1 98 1 ; Bartlett & Nurse 1 990; Nasmyth 1990). START occurs during G 1, regulating the G 1/S transition, and is usually considered the point of commitment to the cell cycle. Before

START

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CELL CYCLE REGULATION

IN YEASTS

237

START, alternative fates are possible, but after START the cell must initiate DNA replication and complete a round of division before other developmental choices are again available. For both fission and budding yeasts, mating is an alternative fate, and for diploids the decision to initiate a meiotic program (sporulation) is also an alternative fate. START-specific mutants were first identified in haploid cells as mutations that blocked the cells in G 1 such that they could, when arrested, undergo mating. Mutants arresting after START are unable to do so. This class of mutations has in budding yeast been termed START I (Reed 1 980). In addition, passage through START determines the rate of progression through the cell cycle. Before passing START, cells must attain a critical cell mass, and the duration of the G1 phase is expandable to allow this. Thus START forms a major rate-limiting step or checkpoint in the cell cycle. Mutations that re­ duce growth rate also delay passage through G 1 , which leads to an accu­ mulation of cells before START. Such lesions have been called START II mutations (Reed 1 980). A priori, it is difficult to distinguish mutations in genes specifically involved in monitoring of the nutritional status of the ceIl (and thus candidates for START regulators) from those in genes with more general effects on growth, which only indirectly influence cell cycle progression. For this reason, we shall only discuss the START I class. The concept of START as a single point of decision, before which all is possible and after which only one thing is possible, is clouded by experi­ mental evidence in the budding yeast in which mutations in some genes are classified as START-specific in haploids but not in diploids (as we will discuss in the section on Gene Functions). Moreover, START is characterized experimentally less by the commitment to division per se, than by the failure to undergo a different fate. The evidence from mutants with START-specific phenotypes in some conditions, but not others, does not suggest that our basic concept of commitment to the cell cycle is in error. Rather it indicates that our concept ofthis commitment as occurring at a single point may be too narrow. Some support for this broadening of START comes from experiments in the fission yeast, which suggest that START-specific gene functions can be separated in time (Novak & Mit­ chison 1 989). START thus may be a window of diminishing options rather than a simple bimodal switch (for a more detailed discussion, see Nurse 1981). Such a window might allow the cell to fine-tune its response to a changing environment and match its alternatives to the circumstances. Reflecting the differently organized cell cycles between the two yeasts, several START genes have been identified in budding yeast, and two in fission yeast. In S. cerevisiae, the most central of these is the CDC28 gene, lesions in which block the cells at the G l iS transition (HartGENE FUNCTIONS

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well et al 1 973; Reed 1 980). However, alleles of CDC28 exist that show a G2 specific block (Piggott et al 1 982; Reed & Wittenberg 1 990), thus revealing a role for this gene function at both points in the cell cycle. CDC28 is a functional and sequence homologue of the S. pombe cdc]+ gene (Beach et al 1 982; Booher & Beach 1 986). Absence of the cdc2+ gene function in fission yeast arrests cells at both the G l /S and G2/M transitions (Nurse & Bissett 1 98 1) . The CDC28/cdc]+ gene encodes a 34-kd serine/ threonine protein kinase (p34Cdc 2; Hindley & Phear 1 984; Simanis & Nurse 1 986; Lorincz & Reed 1 984; Reed et aI 1 985). Moreover, the p 34cdc 2 protein kinase appears to be the linchpin of cell cycle control. In budding yeast, CDC28 appears to act at the interface between signal transduction and G l /S transition (Mendenhall et al 1 987). Recent data (Broek et al 1 99 1 ) suggest that cdc2 i n fission yeast i s a central indicator o f where the cell is in the cycle (see the section on Dependency of S and M). Homologues to the p34cdc2 kinase have been identified in a range of eukaryotes, from fruit flies (Jimenez et al 1 990; Lehner & O'Farrell 1990) to plants (John et al 1 989; Feiler & Jacobs 1 990) to man (Lee & Nurse 1 987; Draetta et al 1 987). Many of these kinases have been demonstrated to be functional homologues: they can replace the p34cdc2 gene function in the yeasts, and thus they must intcract appropriately with both the Gl/S and G2/M controlling networks in both S. cerevisiae and S. pombe (Lee & Nurse 1 987; Wittenberg & Reed 1 989; Jimenez et aI 1 990). This functional conservation strongly argues that at least parts of the controlling networks at the G 1 /S and G2/M boundaries must be conserved in the higher eukaryotes. Other genes at the G 1 /S transition have also been identified in screens for mutations that allow conjugation whcn cells are arrestcd. In S. pombe, lesions in the cdclO+ gene arrest cells before START (Nurse et al 1 976; Aves et aI 1 985). This gene has various sequence elements in common with a rather diverse group of other genes (Avcs et al 1 985); thcse include transcriptional regulators of mating type switching in budding yeast (SW14 and SW16 ) and developmental genes in Drosophila (notch) and C. elegans ( glp 1 and lin 12; Andrews & Herskowitz 1 989; Breeden & Nasmyth 1 987; Yoehem & Greenwald 1 989), all of which share a short block of homology with cdclO+ . The S. cerevisiae cell cycle gene CDC6 shares a different region of homology with cdc10+ (Zhou et al 1 989); however a mutation in CDC6 does not block the cells before START, so it is unclear what significance these homologies have. The cdclO+ gene encodes a phos­ phoprotein of unknown function (Simanis & Nurse 1 989). Numerous genes in budding yeast acting around the G 1 /S transition have now been cloned and characterized. Several of them arrest cells prior to the initiation of DNA replication, but the cells are unable to undergo conjugation. For example, the cdc41S mutation arrests cells beyond the

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ede281S arrest point (Hartwell et al 1 973; Hartwell 1 973), but the cells are unable to mate properly: the conjugation pathway is unavailable to them. However, diploid cells homozygous for ede41s are apparently able to sporu­ late (Hirschenberg & Simchen 1 977), so the meiotic alternative is still open. Thus CDC4 is an important gene in our argument for START as a window (as discussed in the previous section), since it maps after START in haploids and before START in diploids. The CDC7 gene, while not a START gene, is a component of post-START signal transduction; it acts just after CDC4 and encodes a kinase necessary for initiation of DNA replication (Hollingsworth & Sclafani 1 990). Mutations in CDC36 and CDC39 lead to a specific START arrest, but only when the cells are haploid and apparently because the mating type response pathway is activated inappropriately (Neiman et al 1 990; de Barros Lopes et al 1 990). In diploids, edc36ls and cdc39lS cause asynchronous, non-stage specific arrest although confusingly they are required specifically in meiosis (Shuster & Byers 1 989; de Barros Lopes et al 1 990). Where they fit into the START window is unclear. The role of the CDC37 gene, similarly identified as a START function, is likewise uncertain (Reed 1 980). Several new genes have been recently characterized (Prendergast et a1 1 990) including at least one, CDC68, which gives the classic mating-competent START I arrest phenotype. Additional START gene functions have been identified because mutations lead to division at a reduced or increased cell size; the WHIl gene, which will be discussed in detail below, was first isolated in this way (Carter & Sudbery 1 980; Sudbery et al 1 980). The cde281s lesions in budding yeast block the cells most often at G 1 . Suppressor analysis has identified several additional gene functions sur­ rounding CDC28 at START. Three new genes required for progression through START were identified by this approach and were not found in screens for mutations conferring cdc arrest. The first of these, CKSl, is a high copy suppressor of the temperature sensitivity of several cde281s alleles, but is unable to bypass the requirement for CDC28 altogether (Hadwiger et al 1 989a). A deletion of CKSI is lethal and blocks the cells with an unbudded phenotype, indicative of G 1 arrest, although a G2 effect has not been excluded. CKSI encodes a small protein, highly homologous to the sucl + protein of the fission yeast; sucl + has a similar suppressing effect over ede21s mutants, but with effects at the G2/M boundary (dis­ cussed in the section on G2/M Transition). The CKSI protein appears to be associated in a complex with the CDC28 protein kinase, although its precise role is unclear. CLN1 and CLN2, like CKSl, function as high-copy suppressors of cde281s alleles, but are unable to suppress a deletion of CDC28 (Hadwiger et aI 1 989b). These genes have sequence homology to a family of proteins

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called the cyclins, identified first in the marine invertebrates (reviewed by Hunt 1 989); although highly similar to one another, CLNI and CLN2 do not fit into the previously identified classes of A- and B-type cyclins. Sequence comparisons identified another gene in this class of putative G 1 cyclins, which supported the idea that they are a distinct category. This gene was first identified as a dominant mutant allele ( WHIl-l; introduced above), which accelerates the cells through G 1 at a reduced size; later work identified the cyclin homology (Sudbery et al 1 980; Carter & Sudbery 1 980; Nash et al 1 988). Subsequently the same locus was identified in an independent screen, again as a dominant allele (DAFI-l ) , this time isolated because it rendered the cells refractory to G 1 arrest by the mating pher­ omone alpha factor (Cross 1 988). These dominant alleles are truncations of the wild-type protein product (Nash et a1 1 988; Cross 1 988). Deletion of any one or two of the three G I -type cyclins leads to an increase in cell size and delay in G 1 , but is not in itself lethal (Richardson et al 1989). Deletion of all three is lethal, however, and arrests the cells in G 1 (Rich­ ardson et a1 1 989; Cross 1 990). Therefore these three genes are functionally redundant. The WHIlIDAFl gene has also been named CLN3 (Richardson et aI 1 9 89). Overexpression of the wild-type WHIl gene produces a pheno­ type similar to that of the dominant allele DAFl-I: cells are advanced through G 1 at a reduced size (Cross 1 988). Moreover, an activated CLN21 allele, truncated in a manner similar to DAFI-I, has similar dominant effects (Hadwiger et al 1 989b). Thus the G 1 cyclins form one part of a G 1 specific regulatory network that can b e manipulated t o alter the rate of progression through START. A homologue, pucl +, has also been identi­ fied in the fission yeast based on its ability to substitute for WHIl function in budding yeast (S. Forsburg & P. Nurse, in preparation). Confusingly, there are also data suggesting that the WHIl gene plays a role in nutrient signaling not only at the G 1 IS transition, but at mitosis as well (Veinot-Drebot et al 1 990). That there may be roles for the G 1 cyclins at both transitions is further suggested by the isolation of CLNI and CLN2 as high-copy suppressors of a ede28!S allele, which (under certain conditions) has an arrest point just before mitosis (Hadwiger et a1 1 989b; Reed & Wittenberg 1 990). Whether this is due to the partial overlap of cell cycle events in budding yeast, whether it reflects a basic linkage between the role of the kinase at both transitions, or whether it merely suggests non­ specific stabilization of ede28!S by the CLN genes at M remains uncertain. Experiments analogous to those that identified the M block point of this ede28!S allele (Reed & Wittenberg 1 990) may be necessary to conclusively demonstrate the role of the G 1 cyclins. There is a web of connections between the various parts of the cell cycle to ensure that events in parallel pathways

DEPENDENCY OF S UPON M

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occur in an appropriate sequence (Hartwell & Weinert 1 9 89). The depen­ dency of S upon preceding M phase is required to prevent re-replication of the DNA, which would result in polyploidy. There may be little chance of this in a normally growing cell where the staggered timing of the various cycles of DNA replication, spindle organization, mitosis, and budding or septum formation makes overlap unlikely. But if one of these cycles is perturbed, the consequences could be disastrous if the cell were unable to adjust the other cycles. The dependency of S phase upon the preceding M is established at least in part through cdcr in fission yeast as suggested by the results of Broek et al ( 1 99 1 ), who showed that a mutant form of cdc21s can be reset from the G2 to a G 1 form by a heat shock. Such treatment apparently gives the fission yeast cell amnesia; it loses track of its location in the cycle and returns to a pre-S state. These results suggest that substrate availability cannot be the only determinant of cellular memory; if it were, the substrates would still be available when the cdc2+ function recovered. It is possible that association with other proteins d (destroyed by the inactivation of p34c c2) or something innate to the cdc2 protein itself (a temporally regulated modification, for example) accounts for this cellular memory. Therefore, cdc2+ is not merely required for the G 1 IS and G2/M transition, but is changed from one to the next. This change apparently gives the cell memory and helps to establish the proper sequential order between M and S. Although many details are lacking, there is some evidence for a G I-specific protein complex (an S-phase promoting factor, or SPF) between CDC28 and the G 1 cyclins analogous to that we d2 will describe for the G2/M transition. At the later transition, p34c c forms a complex with mitotic eyclins that generates the protein kinase activity required for onset of mitosis. Preliminary evidence suggests that at least CLN2 and, by extrapolation, possibly CLNI and WHI l , are in a complex with CDC28 (Wittenberg et aI 1 990). What is not clear is whether this SPF has protein kinase activity. In vitro kinase activity has not been detected at this stage from S. pombe or higher eukaryotes (Moreno et al 1989), although it has been reported in S. cerevisiae (Reed et a1 1 985; Mendenhall et a1 1 987; Wittenberg & Reed 1 988). More recent data suggest, however, that this S. cerevisiae activity is actually mitotic (Reed & Wittenberg 1 990). This may reflect a difference in substrate specificity in vitro between G 1 /S and G2/M forms of the kinase. The simplest proposal is that the G I cyclins cd 2 associate with p34 c to generate a protein kinase activity appropriate for SPF, which brings about S-phase, but this remains to be fully established. Whatever the precise molecular basis for its action the putative SPF complex must provide an interface with important signals required for G 1 progression. The G I-specific cyclins appear to be periodically regulated,

THE MOLECULAR RESPONSE

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either at the level of mRNA or protein (Wittenberg et al 1 990). Moreover, they are turned over rapidly and, indeed, the dominant alleles CLN2-J, WHIl-J (CLN3-l) and DAFl-J (CLN3-2) are stabilizing truncations that remove so-called PEST sequences that otherwise may target these proteins for prompt degradation (Rogers et a1 1 986; Cross 1 988; Nash et al 1 988; Hadwiger et aI 1 989b). Although functionally redundant, they respond to different signals. CLN2 and WHIl are both modulated at least in part by genes that monitor the mating pheromone response pathway. FARJ is a transcriptional regulator of CLN2 (Chang & Herskowitz 1 990), and FUS3 is a protein kinase that exerts its effect through WHIl (Elion et al 1 990). Both these genes also have effects on the mating pheromone response pathway that are separable from their effects upon the G 1 cyclins. As discussed in the section on Gene Functions, WHIl is implicated in nutrient response at both G l iS and G2/M boundaries (Veinot-Drebot et aI 1 990). It seems likely that a complicated array of signals form the mating pher­ omone response pathway and that the nutrient sensing mechanisms inter­ play to influence the G 1 cyclins, so that while any one of the three responds to a subset of signals, the system as a whole is able to respond appropriately to any changes in the environment.

Is Analysis of the budding yeast meiotic cell cycle has provided some insight into the nature of the requirements for START-specific genes during meiosis. If a control gene were absolutely required to exert some specific effect before DNA replication begins, it should be required for both mitotic and meiotic DNA replication. Such a requirement does not exist for CDC28 (Shuster & Byers 1 989), or even for the somewhat later pre-initiation gene CDC7 (Schild & Byers 1 978). This suggests that START-specific genes exert an indirect effect on the actual onset of DNA replication in mitotic cells and act after DNA replication in meiotic cells. Paradoxically, the CDC36 and CDC39 genes, which fail to give a cell cycle specific arrest in a diploid, are also required at this delayed meiotic START. Perhaps these gene products fulfill several different functions that have yet to be understood. Similar experiments in S. pombe have suggested that cdc10+, but not cdcJ+, is required for initiation of the meiotic program and DNA replication (Beach et aI1985); both these genes map as START genes in the mitotic cycle (Nurse & Bissett 1 98 1 ). One explanation is that meiotic START (as defined by these gene functions) is fundamentally different than mitotic START. However, one would not expect START MEIOTIC G 1

to be completely different in the meiotic pathway, which shares many

processes in common with the mitotic path and which clearly makes use of many genes involved in mitotic progression. Given that expectation, differences between the two are less explicable. Clearly further work is

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required to clarify the similarities and differences of START in mitotic and meiotic cells.

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G2/M Transition If START is the point of commitment to the cell cycle, why is it necessary to have a second regulatory checkpoint later in the cycle? As we have already mentioned, both yeasts have a mitotic control point, but it plays a more prominent role in regulation of the fission yeast cell cycle than in that of the budding yeast. Undergoing mitotis before the completion of S­ phase would be catastrophic for the proper segregation of genetic material, and thus the G2jM transition marks a logical point for a regulatory check­ point. The overall cell size should also be checked, for it may be deleterious to attempt to divide if the cell mass is too small. GENE FUNCTIONS Several genes acting near the G2jM transition have been identified in S. pombe. The p34cdc2 protein kinase, introduced above, it essential for the onset of mitosis in both S. cerevisiae and S. pombe (Nurse & Bissett 1 98 1 ; Piggott et al 1 982; Reed & Wittenberg 1990). Other genes identifying interacting gene functions have been identified by suppressor analysis. Three temperature-sensitive alleles of the previously identified cdc13+ gene (Nurse & Nasmyth 1 98 1 ; Nurse et al 1 976) were isolated as suppressors of a cold-sensitive cdc2 gene, and overproduction of cdcr can rescue the temperature-sensitive allele cdcJ3-117; further, certain allele combinations between cdc2 and cdcJ3 are lethal (Booher & Beach 1 987). The cdc13+ gene is an essential gene in S. pombe, and a deletion of cdcJ3+ block cells at the G2jM transition. However, the conditional point mutation, cdc13-1 17, blocks the cells at the restrictive temperature with a mixed G2jM phenotype in which the chromosomes are condensed, but in which there is an interphase array of microtubules (Nurse et all976; Nasmyth & Nurse 1 98 1 ; Hagan et aI 1 988). cdc13+ has homology to the B-type class of the mitotic cyclins (Solomon et al 1 988; Goebl & Byers 1 988; Hagan et al 1 988; Hunt 1 989). There is evidence for B-type cyclins in the budding yeast as well (K. Nasmyth, personal communication; S. I. Reed, personal communication). The sucJ+ gene was also isolated as a high-copy suppressor of cdc21S (Hayles et al 1 986b) and, moreover, mutations in sucJ+ have been shown to suppress lesions in cdc2 (Hayles et aI 1 986a). �ucJ+ is an essential gene; deletion leads to a cell cycle arrest with a mixed. phenotype and mitotic spindle (Hayles et al 1 986a; Hindley et al 1 987) so that sucl+ appears to be necessary for successful completion of mitosis. The alleles of cdc21S rescued by sucl+ also arrest at the G liS transition, however; so (as pointed out for the G 1 cyclins in S. cerevisiae) the simplest model where sucJ+

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acts only the G2/M transition is not entirely satisfactory. As described in the G l /S Transition section, the S. cerevisiae homologue CKSl was iso­ lated by a similar analysis. Human homologues of this gene have also been identified (Draetta et al 1 987; Richardson et aI 1 990). Additional genes that define a rate-limiting control network acting through cdc2+ have been identified (Figure 2). The cdc25+ gene blocks cells in G2 when mutated (Nurse et aI 1 976). In contrast, overexpression of this gene advances mitosis so that cdc25+ acts as a dosage-dependent inducer of mitosis (Fantes 1 979; Russell & Nurse 1 986). Thus it defines a rate-limit­ ing step in the G2/M transition. A functional homologue of cdc25+ in S. cere­ visiae, MIH1, has been isolated on the basis of its ability to complement the S. pombe mutation (Russell et al 1989). While a deletion of MIHl delays the entry of cells into mitosis, it does not prevent it, and the differences in the controlling network that lead to this effect have not yet been defined. Other cdc25+ homologues have been identified from humans (Sadhu et a1 1 990) and Drosophila (Edgar & O'Farrell 1 989, Jimenez et aI 1 990). Genetic analysis designed specifically to search for rate-limiting genes in S. pombe has identified mutations in which the cells progressed through the cell cycle at a reduced size, as expected if the timing of entry into mitosis is accelerated. These "wee" mutations map to two loci: cdc2+ itself and another gene, weel+ (Nurse 1 975; Nurse & Thuriaux 1980; Fantes 198 1 ) . While a mutation removing weel+ gene function leads to cell division at an abnormally small size, overproduction of wild-type weel + product produces a n elongated cell so that weel+ acts as a dosage-depen­ dent inhibitor of mitosis (Russell & Nurse 1 987a) and thus is opposite in effect to the cdc25+ gene. Although wee celis are accelerated through G2, their G I phase is extended; unlike wild-type cells, wee cells are of insufficient size to pass START when born and must grow to sufficient size during G 1 . A mutation in wee1 suppresses a temperature-sensitive mutation in cdc25. Normally cdc25ts cells are elongated and unable to divide at a high temperature, with a characteristic G2 block, but a lesion in wee1 restores growth to this conditional mutant (Fantes 1 979; Russell & Nurse 1 986). The effects of the two genes are additive, which suggest that they work independently; overexpressing cdc25+ in a weers-deficient background leads to a mitosis that is so accelerated that the cells attempt to divide at too small a size, thus resulting in a disastrous phenomenon known as mitotic catastrophe (see Figure 2). No weel+ homologue has yet been identified in S. cerevisiae, although the S. pombe gene, when overexpressed in the budding yeast, has been demonstrated to have an effect similar to that seen in the fission yeast; the cdc25+ homologue MIH1 can counteract that effect in S. cerevisiae, just as cdc25+ in fission yeast can balance weel + overproduction there (Russell et al 1 989). Thus there

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stf1

cdr2

win1 mcs1-6

additional gene functions (position in pathway not yet completely determined)

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nlm1/cdr1

(kinase) J. wee1 (kinase) 1.

cdc25

.l

regulatory gene functions determining timing of entry into mitosis

wild type (normal phenotype)

Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

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