Sergio Moreno

Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.

Publisher Summary This chapter describes techniques concerned with classical and molecular genetics, cell biology, and biochemistry that can be used with Schizosaccharomyces pombe . Conjugation and sporulation cannot take place in S. pombe except under conditions of nutrient starvation. ME medium is generally used for genetic crosses. To cross two strains, a loopful of h – and a loopful of h + are mixed together on a ME plate. The cross is left to dry and is then incubated below 30°, as conjugation is severely reduced above this temperature. Fully formed four-spore asci can be seen after 2–3 days of incubation. A 2 day-old cross is usually used for tetrad analysis. Using a 3-day-old cross, one can check for the presence of asci under the light microscope. Random spore analysis allows many more spores to be examined than in tetrad analysis, and in this way recombination mapping and strain construction can be carried out. Diploid cells arise spontaneously in most S. pombe strains, this characteristic can be used to isolate homozygous diploids of any strain. For mutagenesis of yeast strains, ethylmethane sulfonate (EMS) and nitrosoguanidine is used.

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.

Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1

Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2 (refs 6–11). Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2 (ref. 8). Here we propose that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. We show by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. We conclude that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA.

Substrates for p34cdc2: In vivo veritas?

Sergio Moreno and Paul Nurse ICRF Cell Cycle Group Microbiology Unit Department of Biochemistry University of Oxford Oxford OX1 3QU England A major reorganization of the cell takes place as it pro- ceeds from interphase to mitosis. The most obvious mi- totic events in the majority of eukaryotic cells are disas- sembly of the nucleus, generation of the mitotic spindle, chromosome condensation, and rounding up of the cell at division. Additionally, organelles present in single or low copy number, such as the Golgi apparatus, fragment into many small components before being partitioned into the two daughter cells. Biochemical processes such as RNA transcription, protein translation, and membrane traffic are also transiently inhibited. At the end of mitosis, these rearrangements and changes are reversed and the cell returns to an interphase state. In eukaryotic cells studied so far, mitotic and meiotic M phase is initiated by activation of the ~34~~~~ protein ki- nase. The activity of this protein kinase rises to a high level at the onset of M phase and remains high throughout most of this process (reviewed in Nurse, 1990). Thus, identifica- tion of the in vivo substrates of the p34* protein kinase is required to understand how the events of mitosis are brought about. Several criteria should be met in order to identify in vivo substrates of a protein kinase. First, the purified protein ki- nase should efficiently phosphorylate the putative sub- strate in vitro. In the case of ~34~~~ it is likely that there will be other structurally similar protein kinases in the cell (Norbury and Nurse, 1989) and so it is important to estab- lish that the reagents and procedures being used to purify the enzyme are adequate to remove all other potentially contaminating protein kinases. Second, the phosphoryla- tion sites in vitro should be identical with the phosphoryla-

Genomic stability and tumour suppression by the APC/C cofactor Cdh1

The anaphase promoting complex or cyclosome (APC/C) is a ubiquitin protein ligase that, together with Cdc20 or Cdh1, targets cell-cycle proteins for degradation. APC/C–Cdh1 specifically promotes protein degradation in late mitosis and G1. Mutant embryos lacking Cdh1 die at E9.5–E10.5 due to defects in the endoreduplication of trophoblast cells and placental malfunction. This lethality is prevented when Cdh1 is expressed in the placenta. Cdh1-deficient cells proliferate inefficiently and accumulate numeric and structural chromosomal aberrations, indicating that Cdh1 contributes to the maintenance of genomic stability. Cdh1 heterozygous animals show increased susceptibility to spontaneous tumours, suggesting that Cdh1 functions as a haploinsufficient tumour suppressor. These heterozygous mice also show several defects in behaviour associated with increased proliferation of stem cells in the nervous system. These results indicate that Cdh1 is required for preventing unscheduled proliferation of specific progenitor cells and protecting mammalian cells from genomic instability.

Conservation of mitotic controls in fission and budding yeasts

In fission yeast, the initiation of mitosis is regulated by a control network that integrates the opposing activities of mitotic inducers and inhibitors. To evaluate whether this control system is likely to be conserved among eukaryotes, we have investigated whether a similar mitotic control operates in the distantly related budding yeast S. cerevisiae. We have found that the protein kinase encoded by the mitotic inhibitor gene wee1+ of fission yeast, which acts to delay mitosis, is able also to delay the initiation of mitosis when expressed in S. cerevisiae. The wee1+ activity is counteracted in S. cerevisiae by the gene product of MIH1, a newly identified gene capable of encoding a protein of MW 54,000, which is a structural and functional homolog of the cdc25+ mitotic inducer of fission yeast. Expression of wee1+ in a mih1- strain prevents the initiation of mitosis. These data indicate that important features of the cdc25+-wee1+ mitotic control network identified in S. pombe are conserved in S. cerevisiae, and therefore are also likely to be generally conserved among eukaryotic organisms.

Regulation of CDK/cyclin complexes during the cell cycle

The eukaryotic cell cycle is regulated by the temporal activation of different cyclin-dependent kinase (CDK)/cyclin complexes. Whilst the level of the catalytic subunit of the complex, the CDK, remains relatively constant through the cycle, the level of the cyclin subunit generally oscillates. Cyclins are synthesized, bind and activate the CDK and are then destroyed. In this review, we summarize the current knowledge of the regulation of the cell cycle by CDK/cyclin complexes with special emphasis on new developments in cyclin biosynthesis and destruction, the structural analysis of the CDK/cyclin complexes and the role of a set of inhibitors of CDK/cyclin complexes that are important for the coordination of the different stages of the cell cycle.

Cross-Talk between Nucleotide Excision and Homologous Recombination DNA Repair Pathways in the Mechanism of Action of Antitumor Trabectedin

Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drugs cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drugs cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death.

Targeting Mitotic Exit Leads to Tumor Regression In Vivo: Modulation by Cdk1, Mastl, and the PP2A/B55α,δ Phosphatase

Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.

Cdh1/Hct1-APC Is Essential for the Survival of Postmitotic Neurons

Cell division at the end of mitosis and G1 is controlled by Cdh1/Hct1, an activator of the E3-ubiquitin ligase anaphase-promoting complex (APC) that promotes the ubiquitylation and degradation of mitotic cyclins and other substrates. Cdh1–APC is active in postmitotic neurons, where it regulates axonal growth and patterning in the developing brain. However, it remains unknown whether Cdh1–APC is involved in preventing cell-cycle progression in terminally differentiated neurons. To address this issue, we used the small hairpin RNA strategy to deplete Cdh1 in postmitotic neurons. We observed that Cdh1 silencing rapidly triggered apoptotic neuronal death. To investigate the underlying mechanism, we focused on cyclin B1, a major Cdh1–APC substrate. Our results demonstrate that Cdh1 is required to prevent the accumulation of cyclin B1 in terminally differentiated neurons. Moreover, by keeping cyclin B1 low, Cdh1 prevented these neurons from entering an aberrant S phase that led to apoptotic cell death. These results provide an explanation for the mechanism of cyclin B1 reactivation that occurs in the brain of patients suffering from neurodegenerative diseases, such as Alzheimers disease.