The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase

1997 ◽  
Vol 110 (13) ◽  
pp. 1523-1531 ◽  
Author(s):  
S. Irniger ◽  
K. Nasmyth
Keyword(s):  
Dna Replication ◽  
Physiological Role ◽  
S Phase ◽  
Yeast Cells ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Cyclin Dependent Kinases ◽  
Initiation Of Dna Replication ◽  
Mutant Cells

Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis. In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle. A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase. The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role. We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication. When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone. DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase. Degradation of Clb2p, Clb3p and Clb5p depends on the APC. This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.

Cdc6p establishes and maintains a state of replication competence during G1 phase

1997 ◽  
Vol 110 (6) ◽  
pp. 753-763 ◽  
Author(s):  
C.S. Detweiler ◽  
J.J. Li
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Transcriptional Repression ◽  
Prolonged Exposure ◽  
S Phase ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Important Intermediate ◽  
Initiation Of Dna Replication ◽  
And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

scholarly journals Cig2, a B-type cyclin, promotes the onset of S in Schizosaccharomyces pombe.

1996 ◽  
Vol 16 (4) ◽  
pp. 1527-1533 ◽  
Author(s):  
O Mondesert ◽  
C H McGowan ◽  
P Russell
Keyword(s):  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Double Mutant ◽  
Kinase Activity ◽  
Nitrogen Starvation ◽  
Gradual Increase ◽  
S Phase ◽  
G1 Arrest ◽  
Cyclin Dependent Kinases ◽  
M Phase

Cdc2, a catalytic subunit of cyclin-dependent kinases, is required for both the G1-to-S and G2-to-M transitions in the fission yeast Schizosaccharomyces pombe. Cdc13, a B-type cyclin, is required for the M-phase induction function of Cd2. Two additional B-type cyclins, Cig1 and Cig2, have been identified in S. pombe, but none of the B-type cyclins are individually required for the onset of S. We report that Cdc13 is important for DNA replication in a strain lacking Cig2. Unlike deltacdc13 cells, double-mutant deltacdc13 deltacig2 cells are defective in undergoing multiple rounds of DNA replication. The conclusion that Cig2 promotes S is further supported by the finding that Cig2 protein and Cig2-associated kinase activity appear soon after the completion of M and peak during S, as well as the observation that S is delayed in deltacig2 cells as they recover from a G1 arrest induced by nitrogen starvation. These studies indicate that Cig2 is the primary S-phase-promoting cyclin in S. pombe but that Cdc13 can effectively substitute for Cig2 in deltacig2 cells. These observations also suggest that the gradual increase in the activity of Cdc2-Cdc13 kinase can be sufficient for the correct temporal ordering of S and M phases in deltacig2 cells.

scholarly journals Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

1999 ◽  
Vol 19 (7) ◽  
pp. 4888-4896 ◽  
Author(s):  
Guy Oshiro ◽  
Julia C. Owens ◽  
Yiqun Shellman ◽  
Robert A. Sclafani ◽  
Joachim J. Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
S Phase ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Initiation Of Dna Replication

ABSTRACT In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

scholarly journals Functional and Physical Interactions between Yeast 14-3-3 Proteins, Acetyltransferases, and Deacetylases in Response to DNA Replication Perturbations

2007 ◽  
Vol 27 (9) ◽  
pp. 3266-3281 ◽  
Author(s):  
Francisca Lottersberger ◽  
Andrea Panza ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Dna Replication ◽  
Histone Acetyltransferase ◽  
S Phase ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Activation ◽  
Histone Tails ◽  
Replicative Stress ◽  
Genotoxic Agents

ABSTRACT The highly conserved 14-3-3 proteins participate in many biological processes in different eukaryotes. The BMH1 and BMH2 genes encode the two functionally redundant Saccharomyces cerevisiae 14-3-3 isoforms. In this work we provide evidence that defective 14-3-3 functions not only impair the ability of yeast cells to sustain DNA replication in the presence of sublethal concentrations of methyl methanesulfonate (MMS) or hydroxyurea (HU) but also cause S-phase checkpoint hyperactivation. Inactivation of the catalytic subunit of the histone acetyltransferase NuA4 or of its interactor Yng2, besides leading to S-phase defects and persistent checkpoint activation in the presence of genotoxic agents, is lethal for bmh mutants. Conversely, the lack of the histone deacetylase subunit Rpd3 or Sin3 partially suppresses the hypersensitivity to HU of bmh mutants and restores their ability to complete DNA replication in the presence of MMS or HU. These data strongly suggest that reduced acetyltransferase functionality might account for the S-phase defects of bmh mutants in the presence of genotoxic agents. Consistent with a role of 14-3-3 proteins in acetyltransferase and deacetylase regulation, we find that acetylation of H3 and H4 histone tails is reduced in temperature-sensitive bmh mutants shifted to the restrictive temperature. Moreover, Bmh proteins physically interact, directly or indirectly, with the Esa1 acetyltransferase throughout the cell cycle and with the Rpd3 deacetylase specifically during unperturbed S phase and after HU treatment. Taken together, our results highlight a novel role for 14-3-3 proteins in the regulation of histone acetyltransferase and deacetylase functions in the response to replicative stress.

scholarly journals Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 550
Author(s):  
Indra A. Shaltiel ◽  
Alba Llopis ◽  
Melinda Aprelia ◽  
Rob Klompmaker ◽  
Apostolos Menegakis ◽  
...  
Keyword(s):  
Dna Damage ◽  
Normal Cell ◽  
S Phase ◽  
G1 Phase ◽  
Anaphase Promoting Complex ◽  
Inhibitory Effects ◽  
Pocket Proteins ◽  
Cyclin Dependent Kinases ◽  
Independent Functions ◽  
Phase Entry

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication

1995 ◽  
Vol 108 (3) ◽  
pp. 927-934 ◽  
Author(s):  
M. Starborg ◽  
E. Brundell ◽  
K. Gell ◽  
C. Larsson ◽  
I. White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Metaphase Chromosomes ◽  
Licensing Factor ◽  
Mammalian Homologue ◽  
Initiation Of Dna Replication

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

Temporal order of DNA replication in the H-2 major histocompatibility complex of the mouse

1992 ◽  
Vol 12 (11) ◽  
pp. 5174-5188
Author(s):  
E G Spack ◽  
E D Lewis ◽  
B Paradowski ◽  
R T Schimke ◽  
P P Jones
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Temporal Order ◽  
Chromosomal Region ◽  
S Phase ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
T Lymphoma Cells ◽  
Initiation Of Dna Replication ◽  
T Lymphoma

As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

scholarly journals Antagonistic regulation of Fus2p nuclear localization by pheromone signaling and the cell cycle

2009 ◽  
Vol 184 (3) ◽  
pp. 409-422 ◽  
Author(s):  
Casey A. Ydenberg ◽  
Mark D. Rose
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Yeast Cells ◽  
Transcriptional Level ◽  
G1 Arrest ◽  
Cycle Arrest ◽  
Pheromone Signaling ◽  
Dependent Inhibition ◽  
Induced Proteins ◽  
Mating Projection

When yeast cells sense mating pheromone, they undergo a characteristic response involving changes in transcription, cell cycle arrest in early G1, and polarization along the pheromone gradient. Cells in G2/M respond to pheromone at the transcriptional level but do not polarize or mate until G1. Fus2p, a key regulator of cell fusion, localizes to the tip of the mating projection during pheromone-induced G1 arrest. Although Fus2p was expressed in G2/M cells after pheromone induction, it accumulated in the nucleus until after cell division. As cells arrested in G1, Fus2p was exported from the nucleus and localized to the nascent tip. Phosphorylation of Fus2p by Fus3p was required for Fus2p export; cyclin/Cdc28p-dependent inhibition of Fus3p during late G1 through S phase was sufficient to block exit. However, during G2/M, when Fus3p was activated by pheromone signaling, Cdc28p activity again blocked Fus2p export. Our results indicate a novel mechanism by which pheromone-induced proteins are regulated during the transition from mitosis to conjugation.

scholarly journals The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function

1999 ◽  
Vol 19 (4) ◽  
pp. 2535-2546 ◽  
Author(s):  
Lynne D. Berry ◽  
Anna Feoktistova ◽  
Melanie D. Wright ◽  
Kathleen L. Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromosome Segregation ◽  
State Level ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Synthetic Lethal ◽  
C Complex ◽  
Mutant Cells

ABSTRACT The Schizosaccharomyces pombe dim1 + gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6mutant. At the restrictive temperature of 36°C, lid1-6mutant cells arrest with a “cut” phenotype similar to that ofcut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein ∼20S complex; the presence of lid1p in this complex depends upon functionalcut9 +. lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity oflid1 +. Further, lid1 +function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.

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