Retinoblastoma protein. Another role rolls in.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
K Nasmyth
Publications and source records attributed to K Nasmyth.
Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast.
In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G1 is necessary for subsequent initiation of DNA replication triggered by activation in late G1 of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6. The absence of pre-RCs during G2 and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G1 depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G1. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G2 and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated. We show that Cdc6p associates with Clb/Cdk1 kinases from late G1 until late anaphase, which might be important for inhibiting pre-RC assembly during S, G2, and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.
Anaphase↗
Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins.
Certain cell types give rise to progeny that adopt different patterns of gene expression in the absence of any differences in their environment. Cells of budding yeast give birth to mother and daughter cells that differ in that only mother cells express the HO endonuclease gene and thereby switch mating types. We describe the identification of five genes, called SHE1-SHE5, that encode cytoplasmic proteins required for mother-specific HO expression. She1p, which is identical to the minimyosin Myo4p, and She3p are not, however, mother-specific proteins. On the contrary, they accumulate in growing buds. She proteins might be required for the transport of factors that promote HO repression from the mother cell into its bud. In an accompanying paper, we show that SHE genes are needed for the accumulation in daughter nuclei of Ash1p, a repressor of HO.
Cell Cycle↗
Asymmetric accumulation of Ash1p in postanaphase nuclei depends on a myosin and restricts yeast mating-type switching to mother cells.
Cell division in haploid yeast gives rise to a "mother" cell capable of mating-type switching and a "daughter" cell that is not. Switching is initiated by the HO endonuclease, whose gene is only transcribed in cells that have previously given birth to a bud (mother cells). HO expression depends on a minimyosin, She1p/Myo4p, which accumulates preferentially in growing buds. We describe a gene, ASH1, that is necessary to repress HO in daughters. ASH1 encodes a zinc finger protein whose preferential accumulation in daughter cell nuclei at the end of anaphase depends on She1p/Myo4p. The greater abundance of Ash1p in daughter cells is responsible for restricting HO expression to mother cells.
Amino Acid Sequence↗
Switching transcription on and off during the yeast cell cycle: Cln/Cdc28 kinases activate bound transcription factor SBF (Swi4/Swi6) at start, whereas Clb/Cdc28 kinases displace it from the promoter in G2.
When yeast cells reach a critical size in late G1 they simultaneously start budding, initiate DNA synthesis, and activate transcription of a set of genes that includes G1 cyclins CLN1, CLN2, and many DNA synthesis genes. Cell cycle-regulated expression of CLN1, CLN2 genes is attributable to the heteromeric transcription factor complex SBF. SBF is composed of Swi4 and Swi6 and binds to the promoters of CLN1 and CLN2. Different cyclin-Cdc28 complexes have different effects on late G1-specific transcription. Activation of transcription at the G1/S boundary requires Cdc28 and one of the G1 cyclins Cln1-Cln3, whereas repression of SBF-regulated genes in G2 requires the association of Cdc28 with G2-specific cyclins Clb1-Clb4. Using in vivo genomic footprinting, we show that SBF (Swi4/Swi6) binding to SCB elements (Swi4/Swi6 cell cycle box) in the CLN2 promoter is cell cycle regulated. SBF binds to the promoter prior to the activation of transcription in late G1, suggesting that Cln/Cdc28 kinase regulates the ability of previously bound SBF to activate transcription. In contrast, SBF dissociates from the CLN2 promoter when transcription is repressed during G2 and M phases, suggesting that Clb1-Clb4 repress SBF activity by inhibiting its DNA-binding activity. Switching transcription on and off by different mechanisms could be important to ensure that Clns are activated only once per cell cycle and could be a conserved feature of cell cycle-regulated transcription.
Bacterial Proteins↗
An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast.
Origins of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle. Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC), which is essential for DNA replication in vivo. Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuclease protection overlapping the ORC footprint. We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo, is essential for the establishment and maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs. These results suggest that pre-RCs play an essential role in initiating DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.
Cell Cycle Proteins↗
At the heart of the budding yeast cell cycle.
It might now seem obvious that the mechanisms regulating cell division would be found to be a highly conserved feature of eukaryotic cells. This was less clear 20 years ago when the pioneering genetic studies of the cell cycle were initiated. This article presents one view as to what lies at the heart of the budding yeast cell cycle. It is written on the premise that most of the key players, such as cyclin-dependent kinases, the anaphase-promoting complex, the origin recognition complex, Cdc6p and Mcm proteins, were performing similar functions in the common ancestor of yeast and man. Ideas about the budding yeast cell cycle might, therefore, have universal significance for other eukaryotic cells.
Anaphase↗
TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast.
The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.
Anaphase↗
The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1.
DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40SIC1 disappears at the G1/S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period.
Base Sequence↗
EGT2 gene transcription is induced predominantly by Swi5 in early G1.
In a screen for cell cycle-regulated genes in the yeast Saccharomyces cerevisiae, we have identified a gene, EGT2, which is involved in cell separation in the G1 stage of the cell cycle. Transcription of EGT2 is tightly regulated in a cell cycle-dependent manner. Transcriptional levels peak at the boundary of mitosis and early G1 The transcription factors responsible for EGT2 expression in early G1 are Swi5 and, to a lesser extent, Ace2. Swi5 is involved in the transcriptional activation of the HO gene during late G1 and early S phase, and Ace2 induces CTS1 transcription during early and late G1 We show that Swi5 activates EGT2 transcription as soon as it enters the nucleus at the end of mitosis in a concentration-dependent manner. Since Swi5 is unstable in the nucleus, its level drops rapidly, causing termination of EGT2 transcription before cells are committed to the next cell cycle. However, Swi5 is still able to activate transcription of HO in late G1 in conjunction with additional activators such as Swi4 and Swi6.
Amino Acid Sequence↗
Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae.
In budding yeast G1 cells increase in cell mass until they reach a critical cell size, at which point (called Start) they enter S phase, bud and duplicate their spindle pole bodies. Activation of the Cdc28 protein kinase by G1-specific cyclins Cln1, Cln2 or Cln3 is necessary for all three Start events. Transcriptional activation of CLN1 and CLN2 by SBF and MBF transcription factors also requires an active Cln-Cdc28 kinase and it has therefore been proposed that the sudden accumulation of CLN1 and CLN2 transcripts during late G1 occurs via a positive feedback loop. We report that whereas Cln1 and Cln2 are required for the punctual execution of most, if not all, other Start-related events, they are not required for the punctual activation of SBF- or MBF-driven transcription. Cln3, on the other hand, is essential. By turning off cyclin B proteolysis and turning on proteolysis of the cyclin B-Cdc28 inhibitor p40SIC1, Cln1 and Cln2 kinases activate cyclin B-Cdc28 kinases and thereby trigger S phase. Thus the accumulation of Cln1 and Cln2 kinases which starts the yeast cell cycle is set in motion by prior activation of SBF- and MBF-mediated transcription by Cln3-Cdc28 kinase. This dissection of regulatory events during late G1 demands a rethinking of Start as a single process that causes cells to be committed to the mitotic cell cycle.
CDC28 Protein Kinase, S cerevisiae↗
Evolution of the cell cycle.
Cell proliferation involves duplication of all cell constituents and their more-or-less equal segregation to daughter cells. It seems probable that the performance of primitive cell-like structures would have been dogged by poor duplication and segregation fidelity, and by parasitism. This favoured evolution of the genome and with it the distinction between 'genomic' components like chromosomes whose synthesis is periodic and most other 'functional' components whose synthesis is continuous. Eukaryotic cells evolved from bacterial ancestors whose fused genome was replicated from a single origin and whose means of segregating sister chromatids depended on fixing their identity at replication. Evolution of an endo- or cytoskeleton, initially as means of consuming other bacteria, eventually enabled evolution of the mitotic spindle and a new means of segregating sister chromatids whose replication could be initiated from multiple origins. In this primitive eukaryotic cell, S and M phases might have been triggered by activation of a single cyclin-dependent kinase whose destruction along with that of other proteins would have triggered anaphase. Mitotic non-disjunction would have greatly facilitated genomic expansion, now possible due to multiple origins, and thereby accelerated the tempo of evolution when permitted by environmental conditions.
Archaea↗
Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae.
S phase entry depends on cyclin-dependent kinases whose activation during late G1 due partly to the synthesis of unstable cyclin subunits. We identify here a second type of unstable protein, Cdc6, whose synthesis during G1 is important for initiation of DNA replication. The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1. The former is due to Swi5, while the latter is due to MBF or SBF transcription factors. Small G1 cells that cannot synthesize Cdc6 in late G1 progress through S phase very slowly. Cells that transcribe CDC6 neither at the end of mitosis nor in late G1 fail to replicate DNA but, despite this, undergo mitosis and produce daughter cells with fractional DNA contents. This 'reductional' anaphase occurs with almost wild-type kinetics and depends on the activity of G2 cyclins. Thus, cells that fail to duplicate chromosomes due to a cdc6 defect cannot prevent the onset of mitosis, unlike other mutants with replication defects. We show, by fluorescence in situ hybridization, that chromosomes which remain unduplicated due to a lack of Cdc6 synthesis are segregated intact to spindle poles during the 'reductional' anaphase.
Anaphase↗
Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast.
The yeast Ste20 protein kinase is involved in pheromone response. Mammalian homologs of Ste20 exist, but their function remains unknown. We identified a novel yeast STE20 homolog, CLA4, in a screen for mutations lethal in the absence of the G1 cyclins Cln1 and Cln2. Cla4 is involved in budding and cytokinesis and interacts with Cdc42, a GTPase required for polarized cell growth. Despite a cytokinesis defect, cla4 mutants are viable. However, double cla4 ste20 mutants cannot maintain septin rings at the bud neck and cannot undergo cytokinesis. Mutations in CDC12, which encodes one of the septins, were found in the same screen. Cla4 and Ste20 kinases apparently share a function in localizing cell growth with respect to the septin ring.
Amino Acid Sequence↗
Cyclin-dependent kinase site-regulated signal-dependent nuclear localization of the SW15 yeast transcription factor in mammalian cells.
Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SWI5 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G1. beta-galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro- Arg-Lys655) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G1 or G2/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser microscopy. Cell cycle-dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser646 and Ser664) to alanine resulted in constitutive nuclear localization. In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS-dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold. We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.
Amino Acid Sequence↗
Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast.
B-type cyclin destruction is necessary for exit from mitosis and the initiation of a new cell cycle. Through the isolation of mutants, we have identified three essential yeast genes, CDC16, CDC23, and CSE1, which are required for proteolysis of the B-type cyclin CLB2 but not of other unstable proteins. cdc23-1 mutants are defective in both entering and exiting anaphase. Their failure to exit anaphase can be explained by defective cyclin proteolysis. CDC23 is required at the metaphase/anaphase transition to separate sister chromatids, and we speculate that it might promote proteolysis of proteins that hold sister chromatids together. Proteolysis of CLB2 is initiated in early anaphase, but a fraction of CLB2 remains stable until anaphase is complete.
Anaphase↗
Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor.
Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae. We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation. Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutation which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo. A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex. A point mutation in the CLB2 UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control of CLB2 transcription. We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.
Base Sequence↗
Terminal differentiation of normal chicken erythroid progenitors: shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control.
Detailed knowledge is available about the molecular makeup of the cell cycle clock in dividing cells. However, comparatively little is known about cell cycle regulation during terminal differentiation. Here we describe a primary cell system in which this question can be addressed. Normal avian erythroid progenitors undergo continuous self-renewal in suspension culture in the presence of growth factors and hormones, allowing us to obtain large cell numbers (10(10)-10(11)). By replacing these "self-renewal factors" with erythropoietin and insulin, the cells can be induced to synchronous, terminal differentiation. During the first 72 h, the cells undergo five cell divisions. Thereafter, they arrest in G1 and complete their maturation into RBC without further divisions. Sixteen to 24 h after induction of differentiation, the cell cycle length decreased from about 20 to 12 h. This shortened doubling time was due to a drastic reduction of G1 (from 12 to 5 h), while S- and G2-phase lengths were not affected. At the same time, the differentiating cells underwent an extensive and concerted switch in their gene expression pattern. During the subsequent four cell divisions, the cell volume decreased from about 300 to less than 70 femtoliters, but the rate of protein synthesis normalized to cell volume remained constant. Interestingly, the shortening of G1 was accompanied by a rapid down-regulation of D-type cyclins and their partner, cyclin-dependent kinase type 4 (cdk4), while expression of S- and G2-M-associated cell cycle regulators (cyclin A and cdk1/cdc2) remained high until the cells arrested in G1 72-96 h after differentiation induction. We conclude that concerted reprogramming of progenitor gene expression during erythroid differentiation is accompanied by profoundly altered cell cycle progression involving the loss or alteration of cell size control at the restriction point.
Animals↗