scholarly journals Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 3315-3322 ◽  
Author(s):  
P A Tavormina ◽  
Y Wang ◽  
D J Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Gamma Irradiation ◽  
Chromosome Segregation ◽  
Mitotic Checkpoint ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dna Metabolism

Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered. We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest. We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints. In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints. Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism. We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore.

scholarly journals Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Christophe de La Roche Saint-André ◽  
Vincent Géli
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genetic Material ◽  
S Phase ◽  
Replication Forks ◽  
H3k4 Methylation ◽  
Origin Firing ◽  
Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

scholarly journals Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (23) ◽  
pp. 10208-10222 ◽  
Author(s):  
Daniel G. Gibson ◽  
Jennifer G. Aparicio ◽  
Fangfang Hu ◽  
Oscar M. Aparicio
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Origin ◽  
S Phase ◽  
Replication Forks ◽  
Cyclin Dependent Kinase ◽  
Chromosomal Dna ◽  
Origin Firing ◽  
Late Origin

ABSTRACT Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Δ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Δ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Δ cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Δ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.

scholarly journals Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
David V Phizicky ◽  
Luke E Berchowitz ◽  
Stephen P Bell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Helicase Loading ◽  
Origin Licensing ◽  
Simultaneous Inhibition ◽  
Mitotic Cells

Meiotic cells undergo a single round of DNA replication followed by two rounds of chromosome segregation (the meiotic divisions) to produce haploid gametes. Both DNA replication and chromosome segregation are similarly regulated by CDK oscillations in mitotic cells. Yet how these two events are uncoupled between the meiotic divisions is unclear. Using Saccharomyces cerevisiae, we show that meiotic cells inhibit both helicase loading and helicase activation to prevent DNA replication between the meiotic divisions. CDK and the meiosis–specific kinase Ime2 cooperatively inhibit helicase loading, and their simultaneous inhibition allows inappropriate helicase reloading. Further analysis uncovered two previously unknown mechanisms by which Ime2 inhibits helicase loading. Finally, we show that CDK and the polo–like kinase Cdc5 trigger degradation of Sld2, an essential helicase–activation protein. Together, our data demonstrate that multiple kinases inhibit both helicase loading and activation between the meiotic divisions, thereby ensuring reductive cell division.

scholarly journals mcl1+, the Schizosaccharomyces pombe Homologue of CTF4, Is Important for Chromosome Replication, Cohesion, and Segregation

2002 ◽  
Vol 1 (5) ◽  
pp. 758-773 ◽  
Author(s):  
Dewight R. Williams ◽  
J. Richard McIntosh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
S Phase ◽  
Sister Chromatids ◽  
Eukaryotic Proteins ◽  
Pulsed Field Electrophoresis ◽  
Phase Progression ◽  
Mutant Cells

ABSTRACT The fission yeast minichromosome loss mutant mcl1-1 was identified in a screen for mutants defective in chromosome segregation. Missegregation of the chromosomes in mcl1-1 mutant cells results from decreased centromeric cohesion between sister chromatids. mcl1+ encodes a β-transducin-like protein with similarity to a family of eukaryotic proteins that includes Ctf4p from Saccharomyces cerevisiae, sepB from Aspergillus nidulans, and AND-1 from humans. The previously identified fungal members of this protein family also have chromosome segregation defects, but they primarily affect DNA metabolism. Chromosomes from mcl1-1 cells were heterogeneous in size or structure on pulsed-field electrophoresis gels and had elongated heterogeneous telomeres. mcl1-1 was lethal in combination with the DNA checkpoint mutations rad3Δ and rad26Δ, demonstrating that loss of Mcl1p function leads to DNA damage. mcl1-1 showed an acute sensitivity to DNA damage that affects S-phase progression. It interacts genetically with replication components and causes an S-phase delay when overexpressed. We propose that Mcl1p, like Ctf4p, has a role in regulating DNA replication complexes.

scholarly journals Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (8) ◽  
pp. 5512-5522 ◽  
Author(s):  
Phuay-Yee Goh ◽  
Uttam Surana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Genetic Interaction ◽  
S Phase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Essential Function ◽  
Additional Role ◽  
Wd40 Repeats

ABSTRACT Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G1/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4(cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1cells. However, the previously characterized mutationcdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G2/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G2/M transition when released from hydroxyurea-induced S-phase arrest. A second function forCDC4 in late S or G2 phase was further confirmed by the observation that cells lacking the CDC4gene are arrested both at G1/S and at G2/M. We subsequently isolated additional temperature-sensitive mutations in theCDC4 gene (such as cdc4-12) that render the mutant defective in both G1/S and G2/M transitions at the restrictive temperature. While the G1/S block in both cdc4-12 and cdc4Δ mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G2/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.

scholarly journals Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Peter E. Burby ◽  
Lyle A. Simmons
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Sos Response ◽  
Bacterial Cells ◽  
Cycle Progression ◽  
Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

scholarly journals Mutations in SID2, a Novel Gene in Saccharomyces cerevisiae, Cause Synthetic Lethality With sic1 Deletion and May Cause a Defect During S Phase

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 17-33
Author(s):  
Matthew D Jacobson ◽  
Claudia X Muñoz ◽  
Kirstin S Knox ◽  
Beth E Williams ◽  
Lenette L Lu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Synthetic Lethality ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Forks ◽  
Novel Gene ◽  
Mutant Strains ◽  
Single Nucleus ◽  
2C Dna Content ◽  
Slow Growing

Abstract SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis. To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1Δ were isolated. In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair. sid2-1 sic1Δ strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an ~2C DNA content. RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1Δ cells. Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest. Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1Δ inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1Δ cells. In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed. sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability. Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

Regulation of Septum Formation in Aspergillus nidulans by a DNA Damage Checkpoint Pathway

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1055-1067
Author(s):  
Steven D Harris ◽  
Peter R Kraus
Keyword(s):  
Dna Damage ◽  
Aspergillus Nidulans ◽  
Cellular Response ◽  
Dna Damage Checkpoint ◽  
Temperature Sensitive ◽  
Dna Metabolism ◽  
Chromosomal Dna ◽  
Checkpoint Response ◽  
Septum Formation ◽  
Checkpoint Pathway

Abstract In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.

scholarly journals The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

2005 ◽  
Vol 168 (7) ◽  
pp. 999-1012 ◽  
Author(s):  
Jeff Bachant ◽  
Shannon R. Jessen ◽  
Sarah E. Kavanaugh ◽  
Candida S. Fielding
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
S Phase ◽  
Origin Of Replication ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Hydroxyurea Treatment ◽  
Chromatid Cohesion ◽  
S Phase Checkpoint

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

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