scholarly journals Budding yeast complete DNA replication after chromosome segregation begins

2018 ◽  
Author(s):  
Tsvetomira Ivanova ◽  
Michael Maier ◽  
Alsu Missarova ◽  
Céline Ziegler-Birling ◽  
Lucas B. Carey ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Normal Growth ◽  
Population Level ◽  
Mitotic Exit ◽  
Yeast Cells ◽  
Cyclin Dependent Kinase ◽  
Temporal Separation ◽  
Entire Genome ◽  
Number Variation

AbstractTo faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in a substantial fraction of unperturbed yeast cells, DNA replication finishes during anaphase, late in mitosis. High cyclin-Cdk activity inhibits replication in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA replication to finish at difficult-to-replicate regions. Replication during late mitosis correlates with elevated mutation rates, including copy number variation. Thus, yeast cells temporally overlap replication and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.One Sentence SummaryCompletion of DNA replication is coupled to downregulation of Cyclin-Dependent Kinase during mitotic exit.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

scholarly journals Bioinformatics Analysis v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Immunoprecipitation, Decross-linking, and DNA Extraction v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

2013 ◽  
Vol 24 (5) ◽  
pp. 578-587 ◽  
Author(s):  
Hui Hua ◽  
Mandana Namdar ◽  
Olivier Ganier ◽  
Juraj Gregan ◽  
Marcel Méchali ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Single Molecule ◽  
Replication Forks ◽  
Cyclin Dependent Kinase ◽  
Chromatin Binding ◽  
Control Factors ◽  
Spindle Disassembly ◽  
Single Molecule Analysis ◽  
Meiosis I

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.

scholarly journals Determine the Size of Sonicated Samples and the DNA Concentration v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Loss of Meiotic Rereplication Block in Saccharomyces cerevisiae Cells Defective in Cdc28p Regulation

2005 ◽  
Vol 4 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Lyndi M. Rice ◽  
Constantine Plakas ◽  
Joseph T. Nickels
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Kinase Inhibitor ◽  
Yeast Cells ◽  
Cyclin Dependent Kinase ◽  
Loss Of Function ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Phosphatase 2A ◽  
Dependent Protein ◽  
Mutant Cells

ABSTRACT Cdc28p is the major cyclin-dependent kinase in Saccharomyces cerevisiae. Its activity is required for blocking the reinitiation of DNA replication during mitosis. Here, we show that under conditions where Cdc28p activity is improperly regulated—either through the loss of function of the Schizosaccharomyces pombe wee1 ortholog Swe1p or through the expression of a dominant CDC28 allele, CDC28AF—diploid yeast cells are able to complete several rounds of premeiotic DNA replication within a single meiotic cell cycle. Moreover, a percentage of mutant cells exhibit a “multispore” phenotype, possessing the ability to package more than four spores within a single ascus. These multispored asci contain both even and odd numbers of viable spores. In order for meiotic rereplication and multispore formation to occur, cells must initiate homologous recombination and maintain proper chromosome cohesion during meiosis I. Rad9p- or Rad17p-dependent checkpoint mechanisms are not required for multispore formation and neither are the B-type cyclin Clb6p and the cyclin-dependent kinase inhibitor Sic1p. Finally, we present evidence of a possible role for a Cdc55p-dependent protein phosphatase 2A in initiating meiotic replication.

scholarly journals An intrinsic S/G2 checkpoint enforced by ATR

Science ◽  
2018 ◽  
Vol 361 (6404) ◽  
pp. 806-810 ◽  
Author(s):  
Joshua C. Saldivar ◽  
Stephan Hamperl ◽  
Michael J. Bocek ◽  
Mingyu Chung ◽  
Thomas E. Bass ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Gene Network ◽  
S Phase ◽  
Genome Integrity ◽  
Control Mechanisms ◽  
Cyclin Dependent Kinase ◽  
G2 Checkpoint ◽  
A Cell ◽  
Early Mitosis

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)–directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.

scholarly journals DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms

2007 ◽  
Vol 27 (14) ◽  
pp. 5067-5078 ◽  
Author(s):  
Fengshan Liang ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Mitotic Exit ◽  
Yeast Cells ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Dna Damage Checkpoints ◽  
Early Anaphase

ABSTRACT Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for “Cdc-fourteen early anaphase release”) and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.

scholarly journals Cell Lysis and Sonication v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

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