scholarly journals RAD9, RAD17, and RAD24 Are Required for S Phase Regulation in Saccharomyces cerevisiae in Response to DNA Damage

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 45-62 ◽  
Author(s):  
A G Paulovich ◽  
R U Margulies ◽  
B M Garvik ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Repair ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Alkylation Damage ◽  
Checkpoint Pathway ◽  
Phase Regulation ◽  
Phase Progression

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9Δ, rad17Δ, and rad24Δ. Furthermore, the triple rad9Δ rad17Δ rad24Δ mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1Δ, mag1Δ, apn1Δ, rev3Δ, rad18Δ, rad16Δ, dun1-Δ100, sad4-1, tel1Δ, rad26Δ, rad51Δ, rad52-1, rad54Δ, rad14Δ, rad1Δ, pol30–46, pol30–52, mad3Δ, pds1Δ/esp2Δ, pms1Δ, mlh1Δ, and msh2Δ are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.

scholarly journals A Role for Saccharomyces cerevisiae Chk1p in the Response to Replication Blocks

2004 ◽  
Vol 15 (9) ◽  
pp. 4051-4063 ◽  
Author(s):  
Kaila L. Schollaert ◽  
Julie M. Poisson ◽  
Jennifer S. Searle ◽  
Jennifer A. Schwanekamp ◽  
Craig R. Tomlinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Transcriptional Response ◽  
S Phase ◽  
Yeast Cells ◽  
Dna Damage Checkpoint ◽  
Checkpoint Kinases ◽  
Checkpoint Pathway ◽  
Dna Replication And Repair ◽  
S Phase Checkpoint

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.

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.

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

2011 ◽  
Vol 366 (1584) ◽  
pp. 3554-3561 ◽  
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.

scholarly journals ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage

2018 ◽  
Vol 115 (7) ◽  
pp. 1523-1528 ◽  
Author(s):  
Dipika Gupta ◽  
Bo Lin ◽  
Ann Cowan ◽  
Christopher D. Heinen
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Alkylating Agent ◽  
Replication Stress ◽  
S Phase ◽  
Dependent Manner ◽  
Alkylation Damage ◽  
Phase Progression ◽  
Dna Alkylating Agent

The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs O6-methylguanine/thymidine (MeG/T) mismatches created upon exposure to DNA alkylating agents. If MeG/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.

scholarly journals Disease-associated DNA2 nuclease–helicase protects cells from lethal chromosome under-replication

2020 ◽  
Author(s):  
Benoît Falquet ◽  
Gizem Ölmezer ◽  
Franz Enkner ◽  
Dominique Klein ◽  
Kiran Challa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Dna Damage Checkpoint ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Essential Function ◽  
Mutant Cells ◽  
Lagging Strand

Abstract DNA2 is an essential nuclease–helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5′-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5′-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2’s role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1’s ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2’s role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.

Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells

DNA Repair ◽  
2008 ◽  
Vol 7 (9) ◽  
pp. 1500-1516 ◽  
Author(s):  
Pierre-Marie Girard ◽  
Mariaelena Pozzebon ◽  
Fabien Delacôte ◽  
Thierry Douki ◽  
Violetta Smirnova ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Checkpoint Response ◽  
Phase Progression ◽  
Functional Dna

scholarly journals Loss of Sin3/Rpd3 Histone Deacetylase Restores the DNA Damage Response in Checkpoint-Deficient Strains of Saccharomyces cerevisiae

2003 ◽  
Vol 23 (13) ◽  
pp. 4522-4531 ◽  
Author(s):  
Kenneth L. Scott ◽  
Sharon E. Plon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Histone Deacetylase ◽  
Dna Damage Response ◽  
Uv Irradiation ◽  
Spindle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Checkpoint Pathway ◽  
Damage Response ◽  
Mutant Strains

ABSTRACT We previously reported that expression of the human forkhead/winged helix transcription factor, CHES1 (checkpoint suppressor 1; FOXN3), suppresses sensitivity to DNA damage and restores damage-induced G2/M arrest in checkpoint-deficient strains of Saccharomyces cerevisiae. We find that a functional glutathione S-transferase-Ches1 fusion protein binds in vivo to Sin3, a component of the S. cerevisiae Sin3/Rpd3 histone deacetylase complex. Checkpoint mutant strains with SIN3 deleted show increased resistance to UV irradiation, which is not further enhanced by CHES1 expression. Conversely, overexpression of SIN3 blocks the Ches1-mediated G2/M delay in response to DNA damage, which is consistent with Ches1 acting by inhibiting the Sin3/Rpd3 complex. Deletion of either SIN3 or RPD3 in rad9 or mec1 checkpoint mutant strains suppresses sensitivity to replication blocks and DNA damage resulting from Cdc9 ligase deficiency and UV irradiation. SIN3 or RPD3 deletions also restored G2/M arrest after DNA damage without concomitant Rad53 phosphorylation in mec1 mutant strains. This DNA damage response is absent in mad1 spindle checkpoint mutants. These data suggest that modulation of chromatin structure may regulate checkpoint responses in S. cerevisiae. Inhibition of histone deacetylation results in a DNA damage checkpoint response mediated by the spindle checkpoint pathway that compensates for loss of the primary DNA damage checkpoint pathway.

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