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PubMed2006

The overexpression of a Saccharomyces cerevisiae centromeric histone H3 variant mutant protein leads to a defect in kinetochore biorientation.

Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA and mediate attachment to the mitotic spindle. Because centromeric sequences are not conserved, centromere identity is propagated by an epigenetic mechanism. All eukaryotes contain an essential histone H3 variant (CenH3) that localizes exclusively to centromeres. Because CenH3 is required for kinetochore assembly and is likely to be the epigenetic mark that specifies centromere identity, it is critical to elucidate the mechanisms that assemble and maintain CenH3 exclusively at centromeres. To learn more about the functions and regulation of CenH3, we isolated mutants in the budding yeast CenH3 that are lethal when overexpressed. These CenH3 mutants fall into three unique classes: (I) those that localize to euchromatin but do not alter kinetochore function, (II) those that localize to the centromere and disrupt kinetochore function, and (III) those that no longer target to the centromere but still disrupt chromosome segregation. We found that a class III mutant is specifically defective in the ability of sister kinetochores to biorient and attach to microtubules from opposite spindle poles, indicating that CenH3 mutants defective in kinetochore biorientation can be obtained.

Cell Cycle Proteins↗
PubMed2006

Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells.

The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis. We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.

Adenomatous Polyposis Coli Protein↗
PubMed2005

Role of Rb family in the epigenetic definition of chromatin.

Epigenetic changes can influence a variety of cellular processes from regulation of gene transcription to proper chromosome segregation. The molecular activities that dictate the assembly, maintenance and regulation of chromatin structure are beginning to be identified. A recent study demonstrates that the Rb family of tumor suppressors plays a major role in global chromatin structure. In addition to the well-known function of Rb family inducing a repressive chromatin state around euchromatic promoters, Rb proteins have a direct role in the assembly of pericentric and telomeric heterochromatin domains. In particular, the Rb family maintains histone 4 lysine 20 tri-methylation (H4K20) at these constitutive heterochromatin domains. Lack of the Rb family results in decreased H4K20 tri-methylation, coincidental with chromosome segregation defects and abnormal telomere elongation, two processes frequently altered in human cancer. Maintenance of heterochromatic domains, such as those of centromeres and telomeres, may represent a novel tumor suppressor function for the Rb family by ensuing genomic stability.

Chromatin Assembly and Disassembly↗
PubMed1986

Follicular maturation, luteinization and first meiotic division in oocytes after inhibiting mitochondrial function in mice with chloramphenicol.

A significant number of diploid oocytes is ovulated from adult NMRI/Han mice treated with high doses of gonadotrophins. This inhibition of the first meiotic division is very likely caused by an altered communication between the germ cell and the surrounding somatic cells leading to a failure of the endocrine control of meiosis. The present study examined the role of mitochondria in follicular development, oocyte maturation and chromosomal segregation during first meiotic division in NMRI/Han mice. To affect mitochondrial function during the late phase of follicular maturation, chloramphenicol, a potent inhibitor of mitochondrial peptidyl transferase, was used. Adult mice were treated with chloramphenicol (CAM; 18.8 or 37.5 mg/kg b.w.) at different times after the pregnant mare serum injection. The results revealed that CAM inhibited the characteristic increase of ovarian weight, reduced the number of oocytes ovulated per female, lowered the progesterone concentration in the postovulatory ovary and increased the incidence of ovulated diploid oocytes. It was concluded that an irregular mitochondrial function may affect normal follicular development and oocyte maturation, and potentially interferes with the order chromosome segregation during the first meiotic division.

Animals↗
PubMed2006

The inhibitor-of-apoptosis protein Bir1p protects against apoptosis in S. cerevisiae and is a substrate for the yeast homologue of Omi/HtrA2.

Inhibitor-of-apoptosis proteins (IAPs) play a crucial role in the regulation of metazoan apoptosis. IAPs are typically characterized by the presence of one to three baculovirus IAP repeat (BIR) domains that are essential for their anti-apoptotic activity. Bir1p is the sole BIR-protein in yeast and has been shown to participate in chromosome segregation events. Here, we show that Bir1p is a substrate for Nma111p, which is the homologue of the human pro-apoptotic serine protease Omi/HtrA2 and which is known to mediate apoptosis in yeast. Bir1p is a cytoplasmic and nuclear protein, and yeast cells lacking bir1 are more sensitive to apoptosis induced by oxidative stress. Consistently, overexpression of Bir1p reduces apoptosis-like cell death, whereas this protective effect can be antagonized in vivo by simultaneous overexpression of Nma111p. Moreover, chronologically aged cells that constitutively overexpress Bir1p show a delayed onset of cell death. Therefore, Bir1p, like its closest metazoan homologues deterin and survivin, has dual functions: it participates in chromosome segregation events and cytokinesis and exhibits anti-apoptotic activity.

Aging↗
PubMed2006

Molecular characterization of teflon, a gene required for meiotic autosome segregation in male Drosophila melanogaster.

Drosophila melanogaster males lack recombination and have evolved a mechanism of meiotic chromosome segregation that is independent of both the chiasmatic and achiasmatic segregation systems of females. The teflon (tef) gene is specifically required in males for proper segregation of autosomes and provides a genetic tool for understanding recombination-independent mechanisms of pairing and segregation as well as differences in sex chromosome vs. autosome segregation. Here we report on the cloning of the tef gene and the molecular characterization of tef mutations. Rescue experiments using a GAL4-driven pUAS transgene demonstrate that tef corresponds to predicted Berkeley Drosophila Genome Project (BDGP) gene CG8961 and that tef expression is required in the male germ line prior to spermatocyte stage S4. Consistent with this early prophase requirement, expression of tef was found to be independent of regulators of meiotic M phase initiation or progression. The predicted Tef protein contains three C2H2 zinc-finger motifs, one at the amino terminus and two in tandem at the carboxyl terminus. In addition to the zinc-finger motifs, a 44- to 45-bp repeat is conserved in three related Drosophila species. On the basis of these findings, we propose a role for Tef as a bridging molecule that holds autosome bivalents together via heterochromatic connections.

Amino Acid Sequence↗
PubMed1996

The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis.

BACKGROUND: Protein phosphatase 2A (PP2A) holoenzymes have a trimeric structure, consisting of a catalytic subunit C and two regulatory subunits A (PR65) and B (PR55). In fission yeast the C subunits, being 80% identical to their mammalian counterparts, are essential for viability and negatively regulate the entry into mitosis. Genetic analyses in budding yeast and Drosophila show that the regulatory subunits are implicated in chromosome segregation, cell morphogenesis and/or cytokinesis. RESULTS: We isolated fission yeast genes paa1+ and pab1+ encoding the regulatory subunits PR65 and PR55, respectively. Gene disruption showed that the paa1+ gene was essential for viability while pab1+ was not required at 26-33 degrees C. Microtubule and actin distributions were anomalous in gene disrupted delta paa1 cells which were incapable of forming a polarized cell shape. Gene disrupted delta pab1 cells were pear- or round-shaped, and lost the polar distributions of actin and microtubules. In addition, delta pab1 cells were defective in cell wall synthesis and sporulation at permissive temperatures. At restrictive temperatures, delta pab1 cells showed an osmoremedial temperature-sensitive phenotype and delayed in cytokinesis. However, chromosome segregation was normal. CONCLUSION: Fission yeast PP2A regulatory subunit plays a critical role in cell morphogenesis, probably through regulation of the cytoskeletal network and cell wall synthesis.

Amino Acid Sequence↗
PubMed2005

Microtubule flux: what is it good for?

During mitosis in a eukaryotic cell, microtubule subunits continuously move towards spindle poles. A new study has revealed that inhibiting this microtubule flux in mammalian cells has no major effects on chromosome movements; it does, however, increase the probability of erroneous chromosome segregation.

Chromosome Segregation↗
PubMed2005

Altered expression of cell cycle genes distinguishes aggressive neuroblastoma.

In this study, gene expression profiling was performed on 103 neuroblastoma (NB) tumors, stages 1-4 with and without MYCN amplification, using cDNA microarrays containing 42,578 elements. Using principal component analysis (PCA) to analyse the relationships among these samples, we confirm that the global patterns of gene expression reflect the phenotype of the tumors. To explore the biological processes that may contribute to increasing aggressive phenotype of the tumors, we utilized a statistical approach based on PCA. We identified a specific subset of the cell cycle and/or chromosome segregation genes that distinguish stage 4 NB tumors from all lower stage tumors, including stage 3. Furthermore, the control of the kinetochore assembly emerges from the Gene Ontology analysis as one of the key biological processes associated with an aggressive NB phenotype. Finally, we establish that these genes are further upregulated in the most aggressive MYCN-amplified tumors.

Cell Cycle↗
PubMed1999

Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe.

Topoisomerases catalyse changes in the topological state of DNA and are required for many aspects of DNA metabolism. While the functions of topoisomerases I and II in eukaryotes are well established, the role of topoisomerase III remains poorly defined. We have identified a gene in the fission yeast Schizosaccharomyces pombe, designated top3 (+), which shows significant sequence similarity to genes encoding topoisomerase III enzymes in other eukaryotic species. In common with murine TOP3 alpha, but in contrast to Saccharomyces cerevisiae TOP3, the S.pombe top3 (+)gene is essential for long-term cell viability. Fission yeast haploid spores containing a disrupted top3 (+)gene germinate successfully, but then undergo only a limited number of cell divisions. Analysis of these top3 mutants revealed evidence of aberrant mitotic chromosome segregation, including the 'cut' phenotype, where septation is completed prior to nuclear division. Consistent with the existence of an intimate association (originally identified in S.cerevisiae ) between topoisomerase III and DNA helicases of the RecQ family, deletion of the rqh1 (+)gene encoding the only known RecQ helicase in S.pombe suppresses lethality in top3 mutants. This conservation of genetic interaction between two widely diverged yeasts suggests that the RecQ family helicases encoded by the Bloom's and Werner's syndrome genes are likely to act in concert with topoisomerase III isozymes in human cells. Our data are consistent with a model in which the association of a RecQ helicase and topoisomerase III is important for facilitating decatenation of late stage replicons to permit faithful chromosome segregation during anaphase.

Amino Acid Sequence↗
PubMed2006

Fission yeast Cut8 is required for the repair of DNA double-strand breaks, ribosomal DNA maintenance, and cell survival in the absence of Rqh1 helicase.

Schizosaccharomyces pombe Rqh1 is a member of the RecQ DNA helicase family. Members of this protein family are mutated in cancer predisposition diseases, causing Bloom's, Werner, and Rothmund-Thomson syndromes. Rqh1 forms a complex with topoisomerase III and is proposed to process or disrupt aberrant recombination structures that arise during S phase to allow proper chromosome segregation during mitosis. Intriguingly, in the absence of Rqh1, processing of these structures appears to be dependent on Rad3 (human ATR) in a manner that is distinct from its role in checkpoint control. Here, we show that rad3 rqh1 mutants are normally committed to a lethal pathway of DNA repair requiring homologous recombination, but blocking this pathway by Rhp51 inactivation restores viability. Remarkably, viability is also restored by overexpression of Cut8, a nuclear envelope protein involved in tethering and proper function of the proteasome. In keeping with a recently described function of the proteasome in the repair of DNA double-strand breaks, we found that Cut8 is also required for DNA double-strand break repair and is essential for proper chromosome segregation in the absence of Rqh1, suggesting that these proteins might function in a common pathway in homologous recombination repair to ensure accurate nuclear division in S. pombe.

Cell Cycle Proteins↗
PubMed2006

Mad2-independent spindle assembly checkpoint activation and controlled metaphase-anaphase transition in Drosophila S2 cells.

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation.

Anaphase↗
PubMed2004

Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast.

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.

Animals↗
PubMed2006

A perikinetochoric ring defined by MCAK and Aurora-B as a novel centromere domain.

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a "cone"-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions.

Animals↗
PubMed2005

Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.

The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

Cell Cycle Proteins↗
PubMed1978

Chromosome age and segregation during sporulation of Bacillus megaterium.

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.

Bacillus megaterium↗
PubMed2000

Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase.

Recently, we cloned two highly related human genes, hChlR1 ( DDX11 ) and hChlR2 ( DDX12 ), which appear to be homologs of the Saccharomyces cerevisiae CHL1 gene. Nucleotide sequence analysis suggests that these genes encode new members of the DEAH family of DNA helicases. While the enzymatic activity of CHL1 has not been characterized, the protein is required for the maintenance of high fidelity chromosome segregation in yeast. Here we report that the hChlR1 protein is a novel human DNA helicase. We have expressed and purified hChlR1 using a baculovirus system and analyzed its enzymatic activity. The recombinant hChlR1 protein possesses both ATPase and DNA helicase activities that are strictly dependent on DNA, divalent cations and ATP. These activities are abolished by a single amino acid substitution in the ATP-binding domain. The hChlR1 protein can unwind both DNA/DNA and RNA/DNA substrates. It has a preference for movement in the 5'-->3' direction on short single-stranded DNA templates. However, unlike other DNA helicases, the hChlR1 DNA helicase can translocate along single-stranded DNA in both directions when substrates have a very long single-stranded DNA region. The enzymatic activities of hChlR1 suggest that DNA helicases are required for maintaining the fidelity of chromosome segregation.

Base Sequence↗
PubMed2006

Cdk and APC activities limit the spindle-stabilizing function of Fin1 to anaphase.

The fidelity of chromosome segregation depends on proper regulation of mitotic spindle behaviour. In anaphase, spindle stability is promoted by the dephosphorylation of cyclin-dependent kinase (Cdk) substrates, which results from Cdk inactivation and phosphatase activation. Few of the critical Cdk targets have been identified. Here, we identify the budding-yeast protein Fin1 (ref. 7) as a spindle-stabilizing protein whose activity is strictly limited to anaphase by changes in its phosphorylation state and rate of degradation. Phosphorylation of Fin1 from S phase to metaphase, by the cyclin-dependent kinase Clb5-Cdk1, inhibits Fin1 association with the spindle. In anaphase, when Clb5-Cdk1 is inactivated, Fin1 is dephosphorylated by the phosphatase Cdc14. Fin1 dephosphorylation targets it to the poles and microtubules of the elongating spindle, where it contributes to spindle integrity. A non-phosphorylatable Fin1 mutant localizes to the spindle before anaphase and impairs efficient chromosome segregation. As cells complete mitosis and disassemble the spindle, the ubiqutin ligase APC(Cdh1) targets Fin1 for destruction. Our studies illustrate how phosphorylation-dependent changes in the behaviour of Cdk1 substrates influence complex mitotic processes.

Anaphase↗