scholarly journals Autorepression of Rfx1 Gene Expression: Functional Conservation from Yeast to Humans in Response to DNA Replication Arrest

2005 ◽  
Vol 25 (23) ◽  
pp. 10665-10673 ◽  
Author(s):  
Yoav Lubelsky ◽  
Nina Reuven ◽  
Yosef Shaul
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Common Mechanism ◽  
Animal Cells ◽  
Replication Checkpoint ◽  
Functional Conservation ◽  
Replication Arrest ◽  
Checkpoint Pathway ◽  
Genes Encoding ◽  
Replication Block

ABSTRACT The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.

scholarly journals Chl12 (Ctf18) Forms a Novel Replication Factor C-Related Complex and Functions Redundantly with Rad24 in the DNA Replication Checkpoint Pathway

2001 ◽  
Vol 21 (17) ◽  
pp. 5838-5845 ◽  
Author(s):  
Takahiro Naiki ◽  
Tae Kondo ◽  
Daisuke Nakada ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Replication ◽  
Dna Damage Checkpoint ◽  
Replication Factor C ◽  
Replication Checkpoint ◽  
Replication Factor ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
Replication Block ◽  
Factor C ◽  
Related Complex

ABSTRACT RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. Therad24Δ mutation enhances the defect ofrfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint.CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Δ norrad24Δ single mutants are defective, chl12Δ rad24Δ double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.

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.

Overlapping Roles of the Spindle Assembly and DNA Damage Checkpoints in the Cell-Cycle Response to Altered Chromosomes in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 521-534
Author(s):  
Peter M Garber ◽  
Jasper Rine
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Spindle Checkpoint ◽  
Dna Lesions ◽  
Replication Proteins ◽  
Replication Checkpoint ◽  
Dna Damage Checkpoints ◽  
Dna Replication Checkpoint ◽  
Mcm Proteins ◽  
Stalled Replication Forks

Abstract The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Δ mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase δ. Thus the specificity of this checkpoint may be more limited than previously recognized.

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 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.

A method for assaying the sensitivity of Drosophila replication checkpoint mutants to anti-cancer and DNA-damaging drugs.

Genome ◽  
2002 ◽  
Vol 45 (5) ◽  
pp. 881-889 ◽  
Author(s):  
Colleen M Radcliffe ◽  
Elizabeth A Silva ◽  
Shelagh D Campbell
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Tumor Formation ◽  
Cycle Progression ◽  
Replication Checkpoint ◽  
Anti Cancer ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes ◽  
Regulate Cell Cycle Progression

In multi-cellular organisms, failure to properly regulate cell-cycle progression can result in inappropriate cell death or uncontrolled cell division leading to tumor formation. To guard against such events, conserved regulatory mechanisms called "checkpoints" block progression into mitosis in response to DNA damage and incomplete replication, as well as in response to other signals. Checkpoint mutants in organisms as diverse as yeast and humans are sensitive to various chemical agents that inhibit DNA replication or cause DNA damage. This phenomenon is the primary rationale for chemotherapy, which uses drugs that preferentially target tumor cells with compromised checkpoints. In this study, we demonstrate the use of Drosophila checkpoint mutants as a system for assaying the effects of various DNA-damaging and anti-cancer agents in a developing multicellular organism. Dwee1, grp and mei-41 are genes that encode kinases that function in the DNA replication checkpoint. We tested zygotic mutants of each gene for sensitivity to the DNA replication inhibitor hydroxyurea (HU), methyl methanosulfonate (MMS), ara-C, cisplatin, and the oxygen radical generating compound paraquat. The mutants show distinct differences in their sensitivity to each of the drugs tested, suggesting an underlying complexity in the responses of individual checkpoint genes to genotoxic stress.Key words: hydroxyurea (HU), ara-C, cisplatin, methyl methane sulfonate (MMS), paraquat.

scholarly journals Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (3) ◽  
pp. 755-764 ◽  
Author(s):  
Tatsushi Wakayama ◽  
Tae Kondo ◽  
Seiko Ando ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Damage ◽  
Cell Growth ◽  
Kinase Activity ◽  
Critical Role ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Replication Checkpoint ◽  
Family Protein ◽  
Replication Block

ABSTRACT In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Δ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Δ cells, as in mec1Δcells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of thepie1Δ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Δ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

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.

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.

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