scholarly journals Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.

1997 ◽  
Vol 8 (8) ◽  
pp. 1587-1601 ◽  
Author(s):  
M R Young ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
S Phase ◽  
Specific Cell ◽  
Replication Origins ◽  
Proliferating Cells ◽  
M Phase ◽  
Mcm Proteins ◽  
And Function

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.

scholarly journals Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (9) ◽  
pp. 5081-5090 ◽  
Author(s):  
M Lei ◽  
Y Kawasaki ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Origins ◽  
Molar Excess ◽  
Physical Interactions ◽  
Mcm Proteins

Mcm2, Mcm3, and Mcm5/Cdc46 are conserved proteins essential for the initiation of DNA synthesis at replication origins in Saccharomyces cerevisiae. The accumulation of these proteins in the nucleus before the onset of DNA synthesis suggests that they play a role in restricting DNA synthesis to once per cell cycle. In this work, we show that Mcm2, Mcm3, and Mcm5 self-interact and interact with one another to form complexes. Mcm2 and Mcm3 are abundant proteins, present in approximately 4 X 10(4) and 2 X 10(5) copies per cell, respectively. Reducing the dosage of Mcm2 by half results in diminished usage of specific replication origins. These results together suggest that a significant molar excess of Mcm proteins relative to replication origins is required for the proper initiation of all replication origins.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

scholarly journals Posttranslational Phosphorylation and Ubiquitination of the Saccharomyces cerevisiae Poly(A) Polymerase at the S/G2 Stage of the Cell Cycle

2000 ◽  
Vol 20 (8) ◽  
pp. 2794-2802 ◽  
Author(s):  
Neptune Mizrahi ◽  
Claire Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Sorting ◽  
S Phase ◽  
Phase Arrest ◽  
Phosphatase Treatment ◽  
M Phase ◽  
64 Kda Protein ◽  
Mitotic Phosphorylation ◽  
Cycle Regulation

ABSTRACT The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA. We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from α-factor-induced G1 arrest or from a hydroxyurea-induced S-phase arrest. While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G2 and disappears by the time cells have reached M phase. The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation. A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment. A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation. However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h. The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems.

scholarly journals Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (12) ◽  
pp. 2858-2864 ◽  
Author(s):  
R K Storms ◽  
R W Ord ◽  
M T Greenwood ◽  
B Mirdamadi ◽  
F K Chu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidylate Synthase ◽  
S Phase ◽  
Mrna Levels ◽  
Activity Profile ◽  
S Period ◽  
Cycle Dependent ◽  
Specific Mrna

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.

scholarly journals Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

2012 ◽  
Vol 287 (15) ◽  
pp. 11891-11898 ◽  
Author(s):  
Kyung Yong Lee ◽  
Sung Woong Bang ◽  
Sang Wook Yoon ◽  
Seung-Hoon Lee ◽  
Jong-Bok Yoon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Replication Origins ◽  
Eukaryotic Chromosome ◽  
M Phase ◽  
Cycle Dependent ◽  
Prereplicative Complex ◽  
Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

scholarly journals Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity

2000 ◽  
Author(s):  
Gideon Grafi ◽  
Brian Larkins
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Storage Protein ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Cyclin B ◽  
Regulatory Subunit ◽  
Maize Endosperm ◽  
M Phase

The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

scholarly journals SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842 ◽  
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (12) ◽  
pp. 2858-2864
Author(s):  
R K Storms ◽  
R W Ord ◽  
M T Greenwood ◽  
B Mirdamadi ◽  
F K Chu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidylate Synthase ◽  
S Phase ◽  
Mrna Levels ◽  
Activity Profile ◽  
S Period ◽  
Cycle Dependent ◽  
Specific Mrna

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.

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