We describe the identification and characterization of the Saccharomyces cerevisiae ZIP2 gene, which encodes a novel meiosis-specific protein essential for synaptonemal complex formation. In the zip2 mutant, chromosomes are homologously paired but not synapsed. The Zip2 protein localizes to discrete foci on meiotic chromosomes; these foci correspond to sites of convergence between paired homologs that are believed to be sites of synapsis initiation. Localization of Zip2p requires the initiation of meiotic recombination. In a mutant defective in double-strand break repair, Zip2p colocalizes with proteins involved in double-strand break formation and processing. We propose that Zip2p promotes the initiation of chromosome synapsis and that localization of Zip2p to sites of interhomolog recombination ensures synapsis between homologous chromosomes.

The hop2 mutant of S. cerevisiae displays a novel phenotype: meiotic chromosomes form nearly wild-type amounts of synaptonemal complex, but most chromosomes are engaged in synapsis with nonhomologous partners. The meiosis-specific Hop2 protein localizes to chromosomes prior to and during synapsis and in the absence of the double-strand breaks that initiate recombination. hop2 strains sustain a wild-type level of meiotic double-strand breaks, but these breaks remain unrepaired. The hop2 mutant arrests at the pachytene stage of meiotic prophase with the RecA-like protein Dmc1 located at numerous sites along synapsed chromosomes. We propose that the Hop2 protein functions to prevent synapsis between nonhomologous chromosomes.

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.entromere-associated protein-E (CENP-E; kinesin-7) is a kinetochore-associated kinesin motor protein with an essential and exclusive role in metaphase chromosome alignment and satisfaction of the mitotic checkpoint (1). CENP-E is a likely candidate to integrate the mechanics of kinetochore-microtubule interaction with the mitotic checkpoint signaling machinery responsible for restraining cell-cycle progression into anaphase. CENP-E is a large dimeric protein consisting of an N-terminal kinesin motor domain tethered to a globular C-terminal domain through an extended coiled-coil rod domain (2, 3). The C-terminal, noncatalytic region of CENP-E is not only sufficient to specify localization to kinetochores, but it also mediates interaction of CENP-E with the serine/threonine kinase BubR1, a key effector of mitotic checkpoint signaling that forms complexes with the checkpoint proteins Cdc20, Bub3, and Mad2 to inhibit the ubiquitin ligase activity of the anaphase promoting complex APC/C CDC20 (4-7). The combined interaction of CENP-E with microtubules and a key regulator of APC/C CDC20 has led to the hypothesis that CENP-E functions as the key kinetochore microtubule receptor responsible for silencing mitotic checkpoint signal transduction after capture of spindle microtubules. This hypothesis was further strengthened by the finding that CENP-E could stimulate the kinase activity of BubR1 in a microtubule-sensitive manner (8, 9). In vitro, the addition of CENP-E to BubR1 resulted in a stimulation of BubR1 kinase activity. The addition of microtubules suppressed this stimulatory activity, an effect thought to be mediated by the CENP-E kinesin motor domain. Although the importance of CENP-E interaction with BubR1 and the role of BubR1-mediated phosphorylation in mitotic checkpoint function remain unclear, CENP-E remains a prominent candidate to play a key role in mitotic checkpoint signal transduction.Depletion of CENP-E from ...

The TAM1 gene of Saccharomyces cerevisiae is expressed specifically during meiosis and encodes a protein that localizes to the ends of meiotic chromosomes. In a taml null mutant, there is an increase in the frequency of chromosomes that fail to recombine and an associated increase in homolog nondisjunction at meiosis I. The taml mutant also displays an increased frequency of precocious separation of sister chromatids and a reduced efficiency of distributive disjunction. The defect in distributive disjunction may be attributable to overloading of the distributive system by the increased number of nonrecombinant chromosomes. Recombination is not impaired in the taml mutant, but crossover interference is reduced substantially. In addition, chromosome synapsis is delayed in taml strains. The combination of a defect in synapsis and a reduction in interference is consistent with previous studies suggesting a role for the synaptonemal complex in regulating crossover distribution, taml is the only known yeast mutant in which the control of crossover distribution is impaired, but the frequency of crossing over is unaffected. We discuss here possibilities for how a telomere-associated protein might function in chromosome synapsis and crossover interference. During meiosis, a single round of DNA duplication is followed by two successive nuclear divisions to generate four haploid progeny from a single diploid cell. The meiosis II division resembles mitotic chromosome segregation , but the meiosis I division is unique in that homologous chromosomes disjoin from each other. A complex series of events unique to prophase of meiosis I ensures that homologs segregate reductionally at the first division. One important aspect of meiotic prophase is a high rate of recombination between homologous chromosomes. Crossing over establishes chiasmata, which are physical connections between homologous chromosomes that persist until metaphase. Chiasmata ensure the proper orientation of chromosomes on the meiosis I spindle and therefore promote reductional chromosome segregation (for review, see Carpenter 1994). The distribution of crossovers, and the chiasmata to which they give rise, is nonrandom in two respects. First, two cross-overs rarely occur closely together-a phenomenon known as crossover interference. Second, every chromosome pair (no matter how small) almost always sustains at least one crossover-referred to as obligate chiasma. A number of studies suggest that crossover interference and obligate chiasma are different manifestations of the 1Corresponding author. E-MAIL: shirleen.roeder@yale.edu; FAX (203) 432-3263. same underlying mechanism (Jones 1967; Kaback et al.

The BDF1 gene of Saccharomyces cerevisiae is required for sporulation. Under starvation conditions, most cells from the bdf1 null mutant fail to undergo one or both meiotic divisions, and there is an absolute defect in spore formation. The Bdf1 protein localizes to the nucleus throughout all stages of the mitotic and meiotic cell cycles. Analysis of spread meiotic nuclei reveals that the Bdf1 protein is localized fairly uniformly along chromosomes, except that it is excluded specifically from the nucleolus. A bdf1 null mutant displays a reduced rate of vegetative growth and sensitivity to a DNA-damaging agent. The BDF1 gene encodes a 77-kDa protein that contains two bromodomains, sequence motifs of unknown function. Separation-of-function alleles suggest that only one of the two bromodomains is required for sporulation, whereas both are required for Bdf1 function in vegetative cells. We propose that the Bdf1 protein is a component of chromatin and that the mitotic and meiotic defects of the bdf1 null mutant result from alterations in chromatin structure.Diploid eukaryotic organisms can undergo two different types of cell division, mitosis and meiosis. During mitosis and the second division of meiosis, sister chromatids separate and segregate, while homologous chromosomes behave independently. In contrast, during the first division of meiosis, homologous chromosomes pair and recombine with each other and then segregate to opposite poles.Although many of the proteins required for a successful meiotic cell cycle are synthesized specifically during meiosis, proteins present in both vegetative and meiotic cells play important roles as well (65). Some proteins serve identical functions during the two types of cell division, but others act differentially. For example, several CDC gene products of Saccharomyces cerevisiae are required for the G 1 -to-S transition in the mitotic cell cycle, but some of these act after DNA replication during meiosis (64). Still other gene products serve functions of different importance in the two types of cell cycle. For instance, genes in the yeast RAD50 series are required for recombinational repair of DNA damage but are otherwise dispensable for vegetative growth (17). However, these genes are essential for the generation of viable meiotic products, because recombination is required for reductional chromosome segregation (53).Chromosomes undergo a dramatic reorganization during prophase I of meiosis I, as homologous chromosomes align with each other to form an elaborate proteinaceous structure called the synaptonemal complex (75). The complex consists of two lateral elements and an intervening central region; each lateral element represents the protein backbone of one pair of condensed sister chromatids. The DNA is organized as a series of chromatin loops, each attached at its base to a lateral element. Not surprisingly, many of the structural components of the synaptonemal complex are synthesized only in meiotic cells (9,27,31,44,69). However, the synaptonemal complex is built upon p...

SummaryIt is now well established that mating can occur between diploid a and a a a a cells of Candida albicans . There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/ a a a a cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.

Cell cycle checkpoints have long been recognized as important nodes for regulating cell proliferation and maintaining genomic integrity. These checkpoints are often altered in cancer and represent promising points for therapeutic intervention. Until recently, direct targeting of the mitotic checkpoint has been an untapped area for cancer drug discovery. Regulation of the mitotic checkpoint is complex, but many of the critical players have been identified and functionally characterized. A substantial number of these proteins can be localized to the kinetochore, a structure located at the centromeric region of each mitotic chromosome. The kinetochore mediates chromosome attachment to spindle microtubules and subsequent chromosome movement. The mitotic checkpoint monitors microtubule attachment and chromosome position on the mitotic spindle, inhibiting progression into anaphase until proper attachment and metaphase positioning is achieved. Centromere-associated protein E is a kinesin microtubule motor protein that plays an essential role in integrating the mechanics of microtubule-chromosome interactions with mitotic checkpoint signaling, and has emerged as a novel target for cancer therapy.

SummaryKinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in 'rigor-like', tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a nonessential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery.