scholarly journals The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (12) ◽  
pp. 2205-2216 ◽  
Author(s):  
Denis Ostapenko ◽  
Janet L. Burton ◽  
Mark J. Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Physiological Role ◽  
S Phase ◽  
G1 Phase ◽  
Deubiquitinating Enzyme ◽  
Anaphase Promoting Complex ◽  
Synthesis Inhibitor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Increased Sensitivity

The anaphase-promoting complex in partnership with its activator, Cdh1, is an E3 ubiquitin ligase responsible for targeting cell cycle proteins during G1 phase. In the budding yeast Saccharomyces cerevisiae, Cdh1 associates with the deubiquitinating enzyme Ubp15, but the significance of this interaction is unclear. To better understand the physiological role(s) of Ubp15, we examined cell cycle phenotypes of cells lacking Ubp15. We found that ubp15∆ cells exhibited delayed progression from G1 into S phase and increased sensitivity to the DNA synthesis inhibitor hydroxyurea. Both phenotypes of ubp15∆ cells were rescued by additional copies of the S-phase cyclin gene CLB5. Clb5 is an unstable protein targeted for proteasome-mediated degradation by several pathways. We found that during G1 phase, the APCCdh1-mediated degradation of Clb5 was accelerated in ubp15∆ cells. Ubp15 interacted with Clb5 independent of Cdh1 and deubiquitinated Clb5 in a reconstituted system. Thus deubiquitination by Ubp15 counteracts APC activity toward cyclin Clb5 to allow Clb5 accumulation and a timely entry into S phase.

scholarly journals Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.

1980 ◽  
Vol 85 (1) ◽  
pp. 108-115 ◽  
Author(s):  
C J Rivin ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cycle Length ◽  
Replication Fork ◽  
Nitrogen Sources ◽  
S Phase ◽  
Phase Duration ◽  
Yeast Saccharomyces Cerevisiae ◽  
Origin Activation ◽  
Cell Cycle Length

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.

Identification of Proteins Whose Synthesis Is Modulated During the Cell Cycle of Saccharomyces cerevisiae

1982 ◽  
Vol 2 (12) ◽  
pp. 1532-1549
Author(s):  
Attila T. Lörincz ◽  
Mark J. Miller ◽  
Nguyen-Huu Xuong ◽  
E. Peter Geiduschek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Radioactive Isotope ◽  
Control Point ◽  
S Phase ◽  
G1 Phase ◽  
Cell Cycle Proteins ◽  
Cell Age ◽  
Synchronous Cell ◽  
Selection Of

We examined the synthesis and turnover of individual proteins in the Saccharomyces cerevisiae cell cycle. Proteins were pulse-labeled with radioactive isotope ( 35 S or 14 C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs. The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, α-factor. In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases. In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation. Based on the time of maximal labeling in the cell cycle and on experiments with α-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase. At least one major control point for the cell cycle proteins occurred between “start” and early S phase. A set of stress-responsive proteins was also identified and analyzed. The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.

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 Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae.

1980 ◽  
Vol 85 (1) ◽  
pp. 96-107 ◽  
Author(s):  
C J Rivin ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cycle Length ◽  
Time Lapse ◽  
S Phase ◽  
Cycle Phase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Emergence ◽  
Length Changes ◽  
Cell Cycle Length

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.

scholarly journals Cell Cycle Progression in G1 and S Phases Is CCR4 Dependent following Ionizing Radiation or Replication Stress in Saccharomyces cerevisiae

2004 ◽  
Vol 3 (2) ◽  
pp. 430-446 ◽  
Author(s):  
Tammy J. Westmoreland ◽  
Jeffrey R. Marks ◽  
John A. Olson ◽  
Eric M. Thompson ◽  
Michael A. Resnick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Ionizing Radiation ◽  
Cell Cycle Progression ◽  
Replication Stress ◽  
S Phase ◽  
Checkpoint Control ◽  
New Genes ◽  
Increased Sensitivity ◽  
Transcription Complexes

ABSTRACT To identify new nonessential genes that affect genome integrity, we completed a screening for diploid mutant Saccharomyces cerevisiae strains that are sensitive to ionizing radiation (IR) and found 62 new genes that confer resistance. Along with those previously reported (Bennett et al., Nat. Genet. 29:426-434, 2001), these genes bring to 169 the total number of new IR resistance genes identified. Through the use of existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4-NOT and RNA polymerase-associated factor 1 (PAF1)-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4Δ cells irradiated in G1 show enhanced lethality compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4Δ cells and CCR4 is a member of the RAD9 epistasis group, thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4Δ cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S-phase replication defect is separate from that seen for rad52 mutants, since rad52Δ ccr4Δ cells show increased sensitivity to HU compared to rad52Δ or ccr4Δ mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4 dependent following radiation or replication stress.

scholarly journals Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (7) ◽  
pp. 3691-3698 ◽  
Author(s):  
D J Burke ◽  
D Church
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Synthesis ◽  
Cell Division ◽  
Cell Separation ◽  
Nuclear Division ◽  
S Phase ◽  
Protein Synthesis Inhibitors ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chemical Inhibitors

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.

Metabolic state and cell cycle as determinants of facilitated uptake of genetic information by yeast Saccharomyces cerevisiae

2008 ◽  
Vol 3 (4) ◽  
pp. 417-421 ◽  
Author(s):  
Larisa Chaustova ◽  
Valė Miliukienė ◽  
Aurelijus Zimkus ◽  
Valdemaras Razumas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Exponential Growth ◽  
Transformation Efficiency ◽  
S Phase ◽  
Yeast Cells ◽  
Yeast Culture ◽  
Metabolic State ◽  
Yeast Saccharomyces Cerevisiae ◽  
Exogenous Dna

AbstractThe transformation efficiency of yeast cells during exponential growth might be characterised as undulatory. The aim of the study was to investigate the reason for the fluctuation in transformation efficiency of yeast Saccharomyces cerevisiae p63-DC5 cells during exponential growth. The heightened response to exogenous DNA was observed with the growing yeast culture when budded cells were predominant. To confirm this phenomenon we carried out synchronization of yeast cells with 10 mM hydroxyurea. Results showed that synchronous yeast cells in the S-phase of cell cycle have enhanced transformation efficiency. Furthermore, S. cerevisiae p63-DC5 cells in the S-phase were successfully transformed with plasmid pl13 in the absence of lithium acetate. We indicated that the permeability of yeast cells in the S-phase to tetraphenylphosphonium (TPP) cations was significantly higher than in asynchronous culture. The results of our study showed that the fluctuation in transformation efficiency was strictly dependent on the metabolic state of yeast cells and the capacity of the yeast cells to become competent was related to the S-phase of cell cycle.

Physical monitoring of mating type switching in Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2342-2349 ◽  
Author(s):  
B Connolly ◽  
C I White ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mating Type ◽  
G1 Phase ◽  
Opposite Mating Type ◽  
Synthesis Inhibitor ◽  
Mating Type Switching ◽  
G2 Phase ◽  
Kinetics Of ◽  
Ho Gene

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.

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