scholarly journals Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

1999 ◽  
Vol 19 (2) ◽  
pp. 1136-1143 ◽  
Author(s):  
Tae Kondo ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Damage ◽  
Genetic Analysis ◽  
The Other ◽  
Amino Terminus ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control ◽  
Checkpoint Pathway ◽  
Two Hybrid

ABSTRACT Genetic analysis has suggested that RAD17,RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast. These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway. Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo. The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by therad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo. Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex. On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1.DDC1 overexpression can partially suppress the phenotypes of the rad24Δ mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53. Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.

scholarly journals Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

2003 ◽  
Vol 23 (4) ◽  
pp. 1441-1452 ◽  
Author(s):  
Vladimir I. Bashkirov ◽  
Elena V. Bashkirova ◽  
Edwin Haghnazari ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Dna Damage ◽  
Target Genes ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Kinase Signaling ◽  
M Cell ◽  
Fha Domain ◽  
Checkpoint Pathway

ABSTRACT The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G2/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G2/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

scholarly journals The Role of Dbf4/Drf1-Dependent Kinase Cdc7 in DNA-Damage Checkpoint Control

2008 ◽  
Vol 32 (6) ◽  
pp. 862-869 ◽  
Author(s):  
Toshiya Tsuji ◽  
Eric Lau ◽  
Gary G. Chiang ◽  
Wei Jiang
Keyword(s):  
Dna Damage ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control

scholarly journals The G(2) DNA damage checkpoint targets both Wee1 and Cdc25

2000 ◽  
Vol 113 (10) ◽  
pp. 1727-1736 ◽  
Author(s):  
J.M. Raleigh ◽  
M.J. O'Connell
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control ◽  
Down Regulation ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Checkpoint Pathway

The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p. Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase. In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G(2) activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation. The protein kinase Chk1p is the endpoint of this checkpoint pathway. We have previously reported that overexpression of Chk1p causes a wee1(+)-dependent G(2) arrest, and this or irradiation leads to hyperphosphorylation of Wee1p. Moreover, Chk1p directly phosphorylates Wee1p in vitro. These data suggested that Wee1p is a key target of Chk1p action in checkpoint control. However, cells lacking wee1(+) are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p. Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest. Chk1p can also phosphorylate Cdc25p in vitro. These phosphorylation events are thought to promote the interaction with 14–3-3 proteins the cytoplasmic retention of the 14–3-3/Cdc25p complexes. However, we show here that the G(2) DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p. Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest. Conversely, inactivation of both wee1(+) and cdc25(+)abolishes checkpoint control. We also show that activation of the G(2) DNA damage checkpoint induces a transient increase in Wee1p levels. We conclude that the G(2) DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability.

scholarly journals The Role of the DNA Damage Checkpoint Pathway in Intraductal Papillary Mucinous Neoplasms of the Pancreas

2007 ◽  
Vol 13 (15) ◽  
pp. 4371-4377 ◽  
Author(s):  
Yoshihiro Miyasaka ◽  
Eishi Nagai ◽  
Hiroshi Yamaguchi ◽  
Kei Fujii ◽  
Takahiro Inoue ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Checkpoint ◽  
Intraductal Papillary Mucinous Neoplasms ◽  
Checkpoint Pathway ◽  
Mucinous Neoplasms

scholarly journals Roles of the Mitotic Inhibitors Wee1 and Mik1 in the G2 DNA Damage and Replication Checkpoints

2001 ◽  
Vol 21 (5) ◽  
pp. 1499-1508 ◽  
Author(s):  
Nicholas Rhind ◽  
Paul Russell
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Dna Damage Checkpoint ◽  
Mitotic Inhibitors

ABSTRACT The G2 DNA damage and DNA replication checkpoints in many organisms act through the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the Wee1/Mik1 family of kinases. However, the in vivo role of these kinases in checkpoint regulation has been unclear. We show that, in the fission yeastSchizosaccharomyces pombe, Mik1 is a target of both checkpoints and that the regulation of Mik1 is, on its own, sufficient to delay mitosis in response to the checkpoints. Mik1 appears to have two roles in the DNA damage checkpoint; one in the establishment of the checkpoint and another in its maintenance. In contrast, Wee1 does not appear to be involved in the establishment of either checkpoint.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
You-hong Wang ◽  
Zhen Guo ◽  
Liang An ◽  
Yong Zhou ◽  
Heng Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nasopharyngeal Cancer ◽  
Noncoding Rnas ◽  
Dependent Protein Kinase ◽  
Damage Repair ◽  
Dependent Protein ◽  
Cancer Tissues

AbstractRadioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

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