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 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 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 The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase

2006 ◽  
Vol 34 (20) ◽  
pp. 5880-5891 ◽  
Author(s):  
Suparna Laha ◽  
Shankar Prasad Das ◽  
Sujata Hajra ◽  
Soumitra Sau ◽  
Pratima Sinha
Keyword(s):  
Dna Damage ◽  
Budding Yeast ◽  
S Phase ◽  
Genome Integrity ◽  
Yeast Protein

scholarly journals Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway

2004 ◽  
Vol 24 (22) ◽  
pp. 10016-10025 ◽  
Author(s):  
Daisuke Nakada ◽  
Yukinori Hirano ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Damage ◽  
Signaling Pathway ◽  
Budding Yeast ◽  
Strand Break ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Mre11 Complex ◽  
Replication Block ◽  
Exonuclease 1 ◽  
Related Proteins

ABSTRACT The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

scholarly journals Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

1997 ◽  
Vol 17 (6) ◽  
pp. 3037-3046 ◽  
Author(s):  
D Pati ◽  
C Keller ◽  
M Groudine ◽  
S E Plon
Keyword(s):  
Dna Damage ◽  
Replication Checkpoint ◽  
Site Analysis ◽  
G2 Checkpoint ◽  
Fork Head ◽  
Checkpoint Pathway ◽  
Dna Binding Site ◽  
Acid Domain ◽  
G2 Delay ◽  
Amino Acid Domain

A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.

scholarly journals Regulation of the cognitive aging process by the transcriptional repressor RP58

2021 ◽  
Author(s):  
Tomoko Tanaka ◽  
Shinobu Hirai ◽  
Hiroyuki Manabe ◽  
Kentaro Endo ◽  
Hiroko Shimbo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Knockout Mice ◽  
Cognitive Aging ◽  
Critical Role ◽  
Harmful Effect ◽  
Transcriptional Repressor ◽  
Repair Pathway ◽  
Wild Type

Aging involves a decline in physiology which is a natural event in all living organisms. An accumulation of DNA damage contributes to the progression of aging. DNA is continually damaged by exogenous sources and endogenous sources. If the DNA repair pathway operates normally, DNA damage is not life threatening. However, impairments of the DNA repair pathway may result in an accumulation of DNA damage, which has a harmful effect on health and causes an onset of pathology. RP58, a zinc-finger transcriptional repressor, plays a critical role in cerebral cortex formation. Recently, it has been reported that the expression level of RP58 decreases in the aged human cortex. Furthermore, the role of RP58 in DNA damage is inferred by the involvement of DNMT3, which acts as a co-repressor for RP58, in DNA damage. Therefore, RP58 may play a crucial role in the DNA damage associated with aging. In the present study, we investigated the role of RP58 in aging. We used RP58 hetero-knockout and wild-type mice in adolescence, adulthood, or old age. We performed immunohistochemistry to determine whether microglia and DNA damage markers responded to the decline in RP58 levels. Furthermore, we performed an object location test to measure cognitive function, which decline with age. We found that the wild-type mice showed an increase in single-stranded DNA and gamma-H2AX foci. These results indicate an increase in DNA damage or dysfunction of DNA repair mechanisms in the hippocampus as age-related changes. Furthermore, we found that, with advancing age, both the wild-type and hetero-knockout mice showed an impairment of spatial memory for the object and increase in reactive microglia in the hippocampus. However, the RP58 hetero-knockout mice showed these symptoms earlier than the wild-type mice did. These results suggest that a decline in RP58 level may lead to the progression of aging.

Role of P53 Mutations in the Radiosensitivity Status of Tumor Cells

1998 ◽  
Vol 84 (5) ◽  
pp. 517-520 ◽  
Author(s):  
Vincenzo Chiarugi ◽  
Lucia Magnelli ◽  
Marina Cinelli
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
P53 Protein ◽  
Cell Cycle Control ◽  
P53 Mutations ◽  
Wild Type ◽  
Bladder Tumors ◽  
Variable Effect ◽  
Wild Type P53

Wild-type p53 is involved in cellular response to DNA damage including cell cycle control, DNA repair and activation of apoptosis. Accumulation of p53 protein following DNA damage may initiate the apoptotic process, resulting in cell death. DNA damage induced by radiation is an example of apoptotic stimulus involving p53. Regulation of apoptosis by p53 can occur through transcriptional regulation of pro-apoptotic (e.g. bax) and anti-apoptotic (e.g. bel-2) factors. Although wild-type p53 usually sensitizes cells to radiation therapy, p53 mutations have a variable effect on radiation response. For example p53 mutations in bone or breast tumors have been found to be associated with resistance to chemotherapeutic drugs or ionizing radiation. Mutated p53 has has been reported to increase sensitivity to radiation and drugs in colorectal and bladder tumors. The present brief commentary tries to find an explanation at molecular level of these conflicting results.

scholarly journals Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

2001 ◽  
Vol 12 (5) ◽  
pp. 1257-1274 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Kazuo Tatebayashi ◽  
Hideo Ikeda ◽  
Ken-ichi Arai ◽  
...  
Keyword(s):  
Dna Replication ◽  
Synthetic Lethality ◽  
S Phase ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Origin Firing ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Pathway ◽  
Nuclear Structures

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect inhsk1-89 is indicated by accumulation ofcut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling.hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those ofrad21-K1 and are significantly enhanced in ahsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

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