scholarly journals Meiotic S-Phase Damage Activates Recombination without Checkpoint Arrest

2005 ◽  
Vol 16 (4) ◽  
pp. 1651-1660 ◽  
Author(s):  
Daniel G. Pankratz ◽  
Susan L. Forsburg
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Meiotic Division ◽  
S Phase ◽  
Wild Type ◽  
Dna Breaks ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Homologous Chromosomes ◽  
Yeast Meiosis

Checkpoints operate during meiosis to ensure the completion of DNA synthesis and programmed recombination before the initiation of meiotic divisions. Studies in the fission yeast Schizosaccharomyces pombe suggest that the meiotic response to DNA damage due to a failed replication checkpoint response differs substantially from the vegetative response, and may be influenced by the presence of homologous chromosomes. The checkpoint responses to DNA damage during fission yeast meiosis are not well characterized. Here we report that DNA damage induced during meiotic S-phase does not activate checkpoint arrest. We also find that in wild-type cells, markers for DNA breaks can persist at least to the first meiotic division. We also observe increased spontaneous S-phase damage in checkpoint mutants, which is repaired by recombination without activating checkpoint arrest. Our results suggest that fission yeast meiosis is exceptionally tolerant of DNA damage, and that some forms of spontaneous S-phase damage can be repaired by recombination without activating checkpoint arrest.

scholarly journals The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage

Cell Division ◽  
2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Muhseena N. Katheeja ◽  
Shankar Prasad Das ◽  
Suparna Laha
Keyword(s):  
Dna Damage ◽  
Budding Yeast ◽  
S Phase ◽  
Wild Type ◽  
Yeast Protein ◽  
Repair Gene ◽  
Replication Checkpoint ◽  
Bud Emergence ◽  
Checkpoint Pathway

Abstract Background The budding yeast protein Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This helicase is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with faster kinetics in chl1 mutants compared to wild-type cells. Also, more damage to DNA is observed in chl1 cells. The viability falls synergistically in rad24chl1 cells. The regulation of Chl1p on budding kinetics in G1 phase falls in line with Rad9p/Chk1p and shows a synergistic effect with Rad24p/Rad53p. rad9chl1 and chk1chl1 shows similar bud emergence as the single mutants chl1, rad9 and chk1. Whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24, rad53 and chl1. In presence of MMS induced damage, synergistic with Rad24p indicates Chl1p’s role as a checkpoint at G1/S acting parallel to damage checkpoint pathway. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further, we have also confirmed that the checkpoint defect functions in parallel to the damage checkpoint pathway of Rad24p. Conclusion Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint activity in presence of damage. This confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1p thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p for Rad53p activation when damaging agents perturb the DNA. Apart from checkpoint activation, it also regulates the budding kinetics as a repair gene.

scholarly journals The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage.

2021 ◽  
Author(s):  
Katheeja Muhseena N. ◽  
Shankar Prasad Das ◽  
Suparna Laha
Keyword(s):  
Dna Damage ◽  
Budding Yeast ◽  
S Phase ◽  
Wild Type ◽  
Yeast Protein ◽  
Replication Checkpoint ◽  
Bud Emergence ◽  
Checkpoint Pathway ◽  
M Phase

Abstract Background: The helicase Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This budding yeast protein is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results: G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with a faster kinetics in chl1 mutants compared to wild-type cells. This role of Chl1p in G1 phase is Rad9p dependent and independent of Rad24 and Rad53. rad9chl1 shows similar bud emergence as the single mutants chl1 and rad9 whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24 , rad53 and chl1 . In case of damage induced by genotoxic agent like hydroxyurea, Chl1p acts as a checkpoint at G1/S. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further we have observed that the checkpoint defect is synergistic with the replication checkpoint Sgs1p and functions in prallel to the checkpoint pathway of Rad24p. Conclusion: Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint, confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1 thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p as well as Rad53p activation when damaging agents perturbs the DNA.

Live observation of fission yeast meiosis in recombination-deficient mutants

2001 ◽  
Vol 114 (15) ◽  
pp. 2843-2853 ◽  
Author(s):  
Monika Molnar ◽  
Jürg Bähler ◽  
Jürg Kohli ◽  
Yasushi Hiraoka
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Meiotic Division ◽  
Chiasma Formation ◽  
Homologous Chromosomes ◽  
Yeast Meiosis ◽  
Mutant Cells ◽  
Random Level ◽  
Meiosis I ◽  
Chromosome Disjunction

Regular segregation of homologous chromosomes during meiotic divisions is essential for the generation of viable progeny. In recombination-proficient organisms, chromosome disjunction at meiosis I generally occurs by chiasma formation between the homologs (chiasmate meiosis). We have studied meiotic stages in living rec8 and rec7 mutant cells of fission yeast, with special attention to prophase and the first meiotic division. Both rec8 and rec7 are early recombination mutants, and in rec7 mutants, chromosome segregation at meiosis I occurs without any recombination (achiasmate meiosis). Both mutants showed distinct irregularities in nuclear prophase movements. Additionally, rec7 showed an extended first division of variable length and with single chromosomes changing back and forth between the cell poles. Two other early recombination deficient mutants (rec14 and rec15) showed very similar phenotypes to rec7 during the first meiotic division, and the fidelity of achiasmate chromosome segregation slightly exceeded the expected random level. We discuss possible regulatory mechanisms of fission yeast to deal with achiasmate chromosome segregation.

scholarly journals Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint

2008 ◽  
Vol 19 (2) ◽  
pp. 445-456 ◽  
Author(s):  
Benoît Froget ◽  
Joël Blaisonneau ◽  
Sarah Lambert ◽  
Giuseppe Baldacci
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Nuclease Activity ◽  
S Phase ◽  
Dna Breaks ◽  
Replication Checkpoint ◽  
Dna Structures ◽  
Replication Arrest ◽  
Dna Replication Checkpoint ◽  
Stalled Fork

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase–specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

scholarly journals Mcl1p Is a Polymerase α Replication Accessory Factor Important for S-Phase DNA Damage Survival

2005 ◽  
Vol 4 (1) ◽  
pp. 166-177 ◽  
Author(s):  
Dewight R. Williams ◽  
J. R. McIntosh
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Synthetic Lethality ◽  
Protein A ◽  
S Phase ◽  
Dynamic Component ◽  
Altered Expression ◽  
Replication Checkpoint ◽  
Replication Arrest ◽  
Accessory Factor

ABSTRACT Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion. The essential function is believed to result from its role as a Pol1p (polymerase α) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p. In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage. Characterization of an inducible allele of mcl1 +, nmt41 mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Δ, rqh1Δ, and hsk1-1312. Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea. We take these data to mean that Mcl1p is a dynamic component of the polymerase α complex during replication and is important for the replication stress response in fission yeast.

scholarly journals DNA Damage and Replication Checkpoints in Fission Yeast Require Nuclear Exclusion of the Cdc25 Phosphatase via 14-3-3 Binding

1999 ◽  
Vol 19 (11) ◽  
pp. 7410-7419 ◽  
Author(s):  
Yan Zeng ◽  
Helen Piwnica-Worms
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Yeast Cells ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Cdc25 Phosphatase ◽  
Checkpoint Response ◽  
Nuclear Exclusion ◽  
Nuclear Location ◽  
Eukaryotic Organisms

ABSTRACT In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.

scholarly journals Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast

2015 ◽  
Vol 26 (8) ◽  
pp. 1440-1451 ◽  
Author(s):  
Heidi M. Blank ◽  
Jason M. Sheltzer ◽  
Colleen M. Meehl ◽  
Angelika Amon
Keyword(s):  
Dna Damage ◽  
S Phase ◽  
Replication Initiation ◽  
Wild Type ◽  
Dna Breaks ◽  
Mitotic Entry ◽  
Dna Replication Initiation ◽  
Yeast Strains ◽  
Similar Phenotype ◽  
Low Levels

Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cell's response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer.

scholarly journals TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus of Tetrahymena

2006 ◽  
Vol 17 (12) ◽  
pp. 5185-5197 ◽  
Author(s):  
J. Sebastian Yakisich ◽  
Pamela Y. Sandoval ◽  
Tara L. Morrison ◽  
Geoffrey M. Kapler
Keyword(s):  
Dna Damage ◽  
Genotoxic Stress ◽  
S Phase ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Strand Breaks ◽  
Origin Binding Protein ◽  
S Phase Checkpoint

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.

scholarly journals The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

2013 ◽  
Vol 24 (21) ◽  
pp. 3350-3357 ◽  
Author(s):  
Tsvetomira Ivanova ◽  
Isabel Alves-Rodrigues ◽  
Blanca Gómez-Escoda ◽  
Chaitali Dutta ◽  
James A. DeCaprio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Yeast Cells ◽  
Replication Checkpoint ◽  
Dna Damaging Agents ◽  
Dna Replication Checkpoint ◽  
Transcriptional Complex ◽  
Carboxy Terminal ◽  
Cycle Regulation

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)–dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

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