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K Nasmyth

Publications and source records attributed to K Nasmyth.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins.

Cohesion between sister chromatids depends on a multisubunit cohesin complex that binds to chromosomes around DNA replication and dissociates from them at the onset of anaphase. Scc2p, though not a cohesin subunit, is also required for sister chromatid cohesion. We show here that Scc2p forms a complex with a novel protein, Scc4p, which is also necessary for sister cohesion. In scc2 or scc4 mutants, cohesin complexes form normally but fail to bind both to centromeres and to chromosome arms. Our data suggest that a major role for the Scc2p/Scc4p complex is to facilitate the loading of cohesin complexes onto chromosomes.

Cell Cycle Proteins↗
PubMed2000

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation.

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.

Anaphase↗
PubMed1999

APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5.

Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome (APC/C) is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases. Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 (APC(Cdh1)). Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1 (refs 4-7). We do not know what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 (APC(Cdc20)) (ref. 4). Here we show that APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae. Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1). Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20.

Anaphase↗
PubMed1999

Identification of cohesin association sites at centromeres and along chromosome arms.

A multisubunit cohesin complex holds sister chromatids together after DNA replication. Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S. cerevisiae. Short DNA sequences (130-280 bp) are sufficient to confer cohesin association. Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p. Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules. Propagation of this structure might be partly epigenetic. Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences.

Binding Sites↗
PubMed1999

Finishing the cell cycle.

The eukaryotic cell division cycle consists of two characteristic states: G1, when replication origins of chromosomes are in a pre-replicative state, and S/G2/M, when they are in a post-replicative state (Nasmyth, 1995). Using straightforward biochemical kinetics, we show that these two states can be created by antagonistic interactions between cyclin-dependent kinases (Cdk) and their foes: the cyclin-degradation machinery (APC) and a stoichiometric inhibitor (CKI). Irreversible transitions between these two self-maintaining steady states drive progress through the cell cycle: at "Start" a cell leaves the G1 state and commences chromosome replication, and at "Finish" the cell separates the products of replication to the incipient daughter cells and re-enters G1. We propose that a protein-phosphatase, by up-regulating the APC and by stabilizing the CKI, plays an essential role at Finish. The phosphatase acts in parallel pathways; hence, cells can leave mitosis in the absence of cyclin degradation or in the absence of the CKI.

Animals↗
PubMed1999

A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis.

A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis. Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II. The eukaryotic cell's cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process.

Anaphase↗
PubMed1999

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.

Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase.

Anaphase↗
PubMed1999

Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

Amino Acid Sequence↗
PubMed1999

Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.

In yeast, anaphase entry depends on Pds1 proteolysis, while chromosome re-duplication in the subsequent S-phase involves degradation of mitotic cyclins such as Clb2. Sequential proteolysis of Pds1 and mitotic cyclins is mediated by the anaphase-promoting complex (APC). Lagging chromosomes or spindle damage are detected by surveillance mechanisms (checkpoints) which block anaphase onset, cytokinesis and DNA re-replication. Until now, the MAD and BUB genes implicated in this regulation were thought to function in a single pathway that blocks APC activity. We show that spindle damage blocks sister chromatid separation solely by inhibiting APCCdc20-dependent Pds1 proteolysis and that this process requires Mad2. Blocking APCCdh1-mediated Clb2 proteolysis and chromosome re-duplication does not require Mad2 but a different protein, Bub2. Our data imply that Mad1, Mad2, Mad3 and Bub1 regulate APCCdc20, whereas Bub2 regulates APCCdh1.

Anaphase↗
PubMed1999

Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex.

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex. An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated. We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains. Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase. DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases.

Amino Acid Sequence↗
PubMed1999

Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter.

Gene activation in eukaryotes requires chromatin remodeling complexes like Swi/Snf and histone acetylases like SAGA. How these factors are recruited to promoters is not yet understood. Using CHIP, we measured recruitment of Swi/Snf, SAGA, the repressor Ash1p, and transcription factors Swi5p and SBF to the HO endonuclease promoter as cells progress through the yeast cell cycle. Swi5p's entry into nuclei at the end of anaphase recruits Swi/Snf, which then recruits SAGA. These two factors then facilitate SBF's binding. Ash1p, which only accumulates in daughter cell nuclei, binds to HO soon after Swi5p and aborts recruitment of Swi/Snf, SAGA, and SBF. Swi5p remains at HO for only 5 min. Swi/Snf's and SAGA's subsequent persistence at HO is self sustaining and constitutes an "epigenetic memory" of HO's transient interaction with Swi5p.

Acetyltransferases↗
PubMed1999

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation.

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1] [2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.

3' Untranslated Regions↗
PubMed1999

Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication.

Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase --> anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a 'Cohesin' complex that binds chromosomes from late G1 until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin's association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G2 and M phases.

Amino Acid Sequence↗
PubMed1999

Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.

Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins.

Blotting, Western↗
PubMed1999

Separating sister chromatids.

Loss of cohesion between sister chromatids triggers their segregation during anaphase. Recent work has identified both a cohesin complex that holds sisters together and a sister-separating protein, separin, that destroys cohesion. Separins are bound by inhibitory proteins whose proteolysis at the metaphase-anaphase transition is mediated by the anaphase-promoting complex and its activator protein CDC20 (APCCDC20). When chromosomes are misaligned, a surveillance mechanism (checkpoint) blocks sister separation by inhibiting APCCDC20. Defects in this apparatus are implicated in causing aneuploidy in human cells.

Anaphase↗
PubMed1998

Model scenarios for evolution of the eukaryotic cell cycle.

Progress through the division cycle of present day eukaryotic cells is controlled by a complex network consisting of (i) cyclin-dependent kinases (CDKs) and their associated cyclins, (ii) kinases and phosphatases that regulate CDK activity, and (iii) stoichiometric inhibitors that sequester cyclin-CDK dimers. Presumably regulation of cell division in the earliest ancestors of eukaryotes was a considerably simpler affair. Nasmyth (1995) recently proposed a mechanism for control of a putative, primordial, eukaryotic cell cycle, based on antagonistic interactions between a cyclin-CDK and the anaphase promoting complex (APC) that labels the cyclin subunit for proteolysis. We recast this idea in mathematical form and show that the model exhibits hysteretic behaviour between alternative steady states: a Gl-like state (APC on, CDK activity low, DNA unreplicated and replication complexes assembled) and an S/M-like state (APC off, CDK activity high, DNA replicated and replication complexes disassembled). In our model, the transition from G1 to S/M ('Start') is driven by cell growth, and the reverse transition ('Finish') is driven by completion of DNA synthesis and proper alignment of chromosomes on the metaphase plate. This simple and effective mechanism for coupling growth and division and for accurately copying and partitioning a genome consisting of numerous chromosomes, each with multiple origins of replication, could represent the core of the eukaryotic cell cycle. Furthermore, we show how other controls could be added to this core and speculate on the reasons why stoichiometric inhibitors and CDK inhibitory phosphorylation might have been appended to the primitive alternation between cyclin accumulation and degradation.

Animals↗