Induction of a G2-Phase Arrest in Xenopus Egg Extracts by Activation of p42 Mitogen-activated Protein Kinase

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G₂-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G₂-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G₂ arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G₂-phase during the first mitotic cell cycle.     

Cdc6 and DNA replication: Limited to humble origins

The budding yeast Cdc6 protein is important for regulating DNA replication intiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc 18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper⁽¹⁾ further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G₁, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.     

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G₁-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G₁-phase. The role of ste9⁺ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G₁-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G₁/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10^ts, cells arrested in G₁-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G₁-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G₁ progression possibly by controlling the amount of the mitotic cyclin in the G₁-phase.     

Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA polymerase α

DNA polymerase α/primase (Polα) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Polα was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Polα and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Polα and by protein affinity chromatography of cell extracts derived from pure G₁-and S-phase cell populations on Polα affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Polα affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Polα (Copeland and Wang, 1991). With 5×10⁸ infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G₁-and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2×10⁹ non synchronous cells, 5×10⁸ G₁-phase cells were isolated. Chromatography of cell extracts derived from G₁-or S-phase cells on Polα affinity columns resulted in identifying several polypeptides in the range of 40–70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G₁-phase extracts.     

Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA polymerase α

DNA polymerase α/primase (Polα) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Polα was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Polα and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Polα and by protein affinity chromatography of cell extracts derived from pure G₁-and S-phase cell populations on Polα affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Polα affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Polα (Copeland and Wang, 1991). With 5×10⁸ infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G₁-and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2×10⁹ non synchronous cells, 5×10⁸ G₁-phase cells were isolated. Chromatography of cell extracts derived from G₁-or S-phase cells on Polα affinity columns resulted in identifying several polypeptides in the range of 40–70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G₁-phase extracts.     

DNA replication in isolated HeLa cell nuclei

DNA replication was investigated in HeLa cell nuclei isolated from different phases of the cell cycle. DNA synthesis occurred only in S-phase nuclei and was dependent on the presence of the four deoxynucleoside triphosphates, Mg⁺⁺, ATP and S-phase cytoplasm. G₁-phase cytoplasm was unable to support such DNA synthesis. A purified preparation of calf thymus DNA polymerase, however, was able to replace S-phase cytoplasm in supporting ATP dependent DNA synthesis, which suggests that the S-phase cytoplasmic factor is a DNA polymerase. G₁-phase nuclei could under no conditions be stimulated to initiate DNA replication prematurely.     

Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells

Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle. Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in higher plants. The complexity represented by different types of CDKs and cyclins in a single species such as alfalfa, indicates that multicomponent regulatory pathways control G₂/M transition. A set of cdc2-related genes (cdc2Ms A, B, D and F) was expressed in G₂ and M cells. Phosphorylation assays also revealed that at least three kinase complexes (Cdc2Ms A/B, D and F) were successively active in G₂/M cells after synchronization. Interaction between alfalfa mitotic cyclin (Medsa;CycB2;1) and a kinase partner has been reported previously. The present yeast two-hybrid analyses showed differential interaction between defined D-type cyclins and Cdc2Ms kinases functioning in G₂/M phases. Localization of Cdc2Ms F kinase to the preprophase band (PPB), the perinuclear ring in early prophase, the mitotic spindle and the phragmoplast indicated a pivotal role for this kinase in mitotic plant cells. So far limited research efforts have been devoted to the functions of phosphatases in the control of plant cell division. A homologue of dual phosphatase, cdc25, has not been cloned yet from alfalfa; however tyrosine phosphorylation was indicated in the case of Cdc2Ms A kinase and the p^13suc1-bound kinase activity was increased by treatment of this complex with recombinant Drosophila Cdc25. The potential role of serine/threonine phosphatases can be concluded from inhibitor studies based on okadaic acid or endothall. Endothall elevated the kinase activity of p13^suc1-bound fractions in G₂-phase alfalfa cells. These biochemical data are in accordance with observed cytological abnormalities. The present overview with selected original data outlines a conclusion that emphasizes the complexity of G₂/M regulatory events in flowering plants.     

The Hect Domain E3 Ligase Tom1 and the F-box Protein Dia2 Control Cdc6 Degradation in G1 Phase

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF^Cdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G₁ phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G₁ phase of the cell cycle and act separately from SCF^Cdc4. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Tom1 and Dia2 each independently immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Tom1 and Dia2 cannot compensate for each other in Cdc6 degradation. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G₁ phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G₁ that may regulate pre-replicative complex assembly.     

A WD Repeat Protein Controls the Cell Cycle and Differentiation by Negatively Regulating Cdc2/B-Type Cyclin Complexes

In the fission yeast Schizosaccharomyces pombe, p34^cdc2 plays a central role controlling the cell cycle. We recently isolated a new gene named srw1⁺, capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34^cdc2. Cells lacking srw1⁺ are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G₁ and die of accelerated mitotic catastrophe if regulation of p34^cdc2/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34^cdc2. srw1⁺ shares functional similarity with rum1⁺, having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34^cdc2 and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.     

A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea

By incubating at 30°C in the presence of an energy source, p34^cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40°C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34^cdc2/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34^cdc2/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40°C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34^cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34^cdc2/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34^cdc2/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34^cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34^cdc2/cyclin B, which initiates mitosis through the activation of Cdc25C.     

Functional analysis of subcellular localization and protein-protein interaction sequences in the essential DNA ligase I protein of fission yeast

DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear–mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box. Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase.     

SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.

Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. Genetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/CDC28. As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE-fractionated yeast extract. Multiubiquitination depends on cyclin/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient to serve as substrate for CDC34-dependent ubiquitination. The complementary C-terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1.     

Consequences of abnormal CDK activity in S phase

Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1^? strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.     

Basal-Cell Subpopulations and Cell-Cycle Kinetics In Human Epidermal Explant Cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different ³H-thymidine (³H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that ³H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G₂-phase cells were cycling, whereas some 20% of the cells stayed in G₁-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G₁- and G₂-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G₂-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated ³H-dThd at a higher rate than cells with high RNA contents.     

PP2ACdc55 regulates G1 cyclin stability

Maintaining accurate progression through the cell cycle requires the proper temporal expression and regulation of cyclins. The mammalian D-type cyclins promote G₁-S transition. D1 cyclin protein stability is regulated through its ubiquitylation and resulting proteolysis catalyzed by the SCF E3 ubiquitin ligase complex containing the F-box protein, Fbx4. SCF E3-ligase-dependent ubiquitylation of D1 is trigged by an increase in the phosphorylation status of the cyclin. As inhibition of ubiquitin-dependent D1 degradation is seen in many human cancers, we set out to uncover how D-type cyclin phosphorylation is regulated. Here we show that in S. cerevisiae, a heterotrimeric protein phosphatase 2A (PP2A^Cdc55) containing the mammalian PPP2R2/PR55 B subunit ortholog Cdc55 regulates the stability of the G₁ cyclin Cln2 by directly regulating its phosphorylation state. Cells lacking Cdc55 contain drastically reduced Cln2 levels caused by degradation due to cdk-dependent hyperphosphorylation, as a Cln2 mutant unable to be phosphorylated by the yeast cdk Cdc28 is highly stable in cdc55-null cells. Moreover, cdc55-null cells become inviable when the SCF^Grr1 activity known to regulate Cln2 levels is eliminated or when Cln2 is overexpressed, indicating a critical relationship between SCF and PP2A functions in regulating cell cycle progression through modulation of G₁-S cyclin degradation/stability. In sum, our results indicate that PP2A is absolutely required to maintain G₁-S cyclin levels through modulating their phosphorylation status, an event necessary to properly transit through the cell cycle.     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF^CDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a β-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.