Stochasticity Triggers Activation of the S-phase Checkpoint Pathway in Budding Yeast

Replication blocks and DNA damage incurred during S phase activate the S-phase and intra-S-phase checkpoint responses, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1Δ cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1Δ dun1Δ cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

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Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway

Genes & Development ◽

10.1101/gad.12.18.2956 ◽

1998 ◽

Vol 12 (18) ◽

pp. 2956-2970 ◽

Cited By ~ 277

Author(s):

B. A. Desany ◽

A. A. Alcasabas ◽

J. B. Bachant ◽

S. J. Elledge

Keyword(s):

S Phase ◽

Checkpoint Pathway ◽

Essential Function ◽

S Phase Checkpoint

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S-Phase Checkpoint Pathways Stimulate the Mobility of the Retrovirus-Like Transposon Ty1

Molecular and Cellular Biology ◽

10.1128/mcb.01095-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8874-8885 ◽

Cited By ~ 29

Author(s):

M. Joan Curcio ◽

Alison E. Kenny ◽

Sharon Moore ◽

David J. Garfinkel ◽

Matthew Weintraub ◽

...

Keyword(s):

Dna Damage ◽

Reverse Transcriptase ◽

Reverse Transcriptase Activity ◽

S Phase ◽

Dna Lesions ◽

Restriction Factors ◽

Transcriptase Activity ◽

Checkpoint Pathway ◽

Ty1 Mobility ◽

S Phase Checkpoint

ABSTRACT The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101Δ mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101Δ mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

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

Cell Division ◽

10.1186/s13008-021-00072-x ◽

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.

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

10.21203/rs.3.rs-151524/v1 ◽

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.

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Surviving chromosome replication: the many roles of the S-phase checkpoint pathway

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0071 ◽

2011 ◽

Vol 366 (1584) ◽

pp. 3554-3561 ◽

Cited By ~ 65

Author(s):

Karim Labib ◽

Giacomo De Piccoli

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Cell Cycle Progression ◽

Reductase Activity ◽

Critical Role ◽

Chromosome Replication ◽

S Phase ◽

Replication Forks ◽

Checkpoint Pathway ◽

S Phase Checkpoint

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.

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Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

Nucleic Acids Research ◽

10.1093/nar/gkl741 ◽

2006 ◽

Vol 34 (20) ◽

pp. 5852-5862 ◽

Cited By ~ 18

Author(s):

Violeta Cordón-Preciado ◽

Sandra Ufano ◽

Avelino Bueno

Keyword(s):

Budding Yeast ◽

S Phase ◽

Dna Alkylation ◽

Alkylation Damage ◽

S Phase Checkpoint

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Inhibition of spindle extension through the yeast S phase checkpoint is coupled to replication fork stability and the integrity of centromeric DNA

Molecular Biology of the Cell ◽

10.1091/mbc.e19-03-0156 ◽

2019 ◽

Vol 30 (22) ◽

pp. 2771-2789

Author(s):

Jeff Julius ◽

Jie Peng ◽

Andrew McCulley ◽

Chris Caridi ◽

Remigiusz Arnak ◽

...

Keyword(s):

Mitotic Spindle ◽

Replication Fork ◽

Budding Yeast ◽

S Phase ◽

Force Balance ◽

Replication Forks ◽

Origin Firing ◽

Spindle Structure ◽

Kinetochore Function ◽

S Phase Checkpoint

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

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