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 Replication Fork Slowing and Stalling are Distinct, Checkpoint-Independent Consequences of Replicating Damaged DNA

2017 ◽  
Author(s):  
Divya Ramalingam Iyer ◽  
Nicholas Rhind
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Single Molecule ◽  
Replication Fork ◽  
Replication Forks ◽  
Origin Firing ◽  
Dna Combing ◽  
Fork Stalling

AbstractIn response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.Author SummaryFaithful duplication of the genome is essential for genetic stability of organisms and species. To ensure faithful duplication, cells must be able to replicate damaged DNA. To do so, they employ checkpoints that regulate replication in response to DNA damage. However, the mechanisms by which checkpoints regulate DNA replication forks, the macromolecular machines that contain the helicases and polymerases required to unwind and copy the parental DNA, is unknown. We have used DNA combing, a single-molecule technique that allows us to monitor the progression of individual replication forks, to characterize the response of fission yeast replication forks to DNA damage that blocks the replicative polymerases. We find that forks pass most lesions with only a brief pause and that this lesion bypass is checkpoint independent. However, at a low frequency, forks stall at lesions, and that the checkpoint is required to prevent these stalls from accumulating single-stranded DNA. Our results suggest that the major role of the checkpoint is not to regulate the interaction of replication forks with DNA damage, per se, but to mitigate the consequences of fork stalling when forks are unable to successfully navigate DNA damage on their own.

scholarly journals Critical role of SMG7 in activation of the ATR-CHK1 axis in response to genotoxic stress

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathleen Ho ◽  
Hongwei Luo ◽  
Wei Zhu ◽  
Yi Tang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Genotoxic Stress ◽  
Dna Damage Checkpoint ◽  
Cycle Progression ◽  
Essential Step

AbstractCHK1 is a crucial DNA damage checkpoint kinase and its activation, which requires ATR and RAD17, leads to inhibition of DNA replication and cell cycle progression. Recently, we reported that SMG7 stabilizes and activates p53 to induce G1 arrest upon DNA damage; here we show that SMG7 plays a critical role in the activation of the ATR-CHK1 axis. Following genotoxic stress, SMG7-null cells exhibit deficient ATR signaling, indicated by the attenuated phosphorylation of CHK1 and RPA32, and importantly, unhindered DNA replication and fork progression. Through its 14-3-3 domain, SMG7 interacts directly with the Ser635-phosphorylated RAD17 and promotes chromatin retention of the 9-1-1 complex by the RAD17-RFC, an essential step to CHK1 activation. Furthermore, through maintenance of CHK1 activity, SMG7 controls G2-M transition and facilitates orderly cell cycle progression during recovery from replication stress. Taken together, our data reveals SMG7 as an indispensable signaling component in the ATR-CHK1 pathway during genotoxic stress response.

scholarly journals Mechanism of Caffeine-Induced Checkpoint Override in Fission Yeast

2000 ◽  
Vol 20 (12) ◽  
pp. 4288-4294 ◽  
Author(s):  
Bettina A. Moser ◽  
Jean-Marc Brondello ◽  
Beth Baber-Furnari ◽  
Paul Russell
Keyword(s):  
Dna Damage ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Protein Kinases ◽  
Molecular Target ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Mitotic Checkpoints ◽  
Direct Activation

ABSTRACT Mitotic checkpoints restrain the onset of mitosis (M) when DNA is incompletely replicated or damaged. These checkpoints are conserved between the fission yeast Schizosaccharomyces pombe and mammals. In both types of organisms, the methylxanthine caffeine overrides the synthesis (S)-M checkpoint that couples mitosis to completion of DNA S phase. The molecular target of caffeine was sought in fission yeast. Caffeine prevented activation of Cds1 and phosphorylation of Chk1, two protein kinases that enforce the S-M checkpoint triggered by hydroxyurea. Caffeine did not inhibit these kinases in vitro but did inhibit Rad3, a kinase that regulates Cds1 and Chk1. In accordance with this finding, caffeine also overrode the G2-M DNA damage checkpoint that requires Rad3 function. Rad3 coprecipitated with Cds1 expressed at endogenous amounts, a finding that supports the hypothesis that Rad3 is involved in direct activation of Cds1.

scholarly journals Monitoring Schizosaccharomyces pombe genome stress by visualizing end-binding protein Ku

Biology Open ◽  
2021 ◽  
Vol 10 (2) ◽  
Author(s):  
Chance E. Jones ◽  
Susan L. Forsburg
Keyword(s):  
Dna Damage ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Protein Complex ◽  
Genome Stability ◽  
Binding Protein ◽  
Dna Lesions ◽  
Live Cells ◽  
Dna Damage Checkpoint ◽  
Ku Protein

ABSTRACT Studies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

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 Schizosaccharomyces pombe Assays to Study Mitotic Recombination Outcomes

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 79
Author(s):  
Hannah M. Hylton ◽  
Bailey E. Lucas ◽  
Ruben C. Petreaca
Keyword(s):  
Dna Damage ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Molecular Mechanisms ◽  
Mitotic Recombination ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Repair

The fission yeast—Schizosaccharomyces pombe—has emerged as a powerful tractable system for studying DNA damage repair. Over the last few decades, several powerful in vivo genetic assays have been developed to study outcomes of mitotic recombination, the major repair mechanism of DNA double strand breaks and stalled or collapsed DNA replication forks. These assays have significantly increased our understanding of the molecular mechanisms underlying the DNA damage response pathways. Here, we review the assays that have been developed in fission yeast to study mitotic recombination.

scholarly journals GTSE1 is possibly involved in the DNA damage repair and cisplatin resistance in osteosarcoma

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Chaofan Xie ◽  
Wei Xiang ◽  
Huiyong Shen ◽  
Jingnan Shen
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Cisplatin Resistance ◽  
Dna Damage Repair ◽  
Loss Of Function ◽  
Damage Repair

Abstract Background G2 and S phase-expressed-1 (GTSE1) negatively regulates the tumor-suppressive protein p53 and is potentially correlated with chemoresistance of cancer cells. This study aims to explore the effect of GTSE1 on the DNA damage repair and cisplatin (CDDP) resistance in osteosarcoma (OS). Materials and methods Expression of GTSE1 in OS was predicted in bioinformatics system GEPIA and then validated in clinically obtained tissues and acquired cell lines using RT-qPCR, immunohistochemical staining, and western blot assays. Gain- and loss-of-function studies of GTSE1 were performed in MG-63 and 143B cells to examine its function in cell cycle progression, DNA replication, and CDDP resistance. Stably transfected MG-63 cells were administrated into mice, followed by CDDP treatment to detect the role of GTSE1 in CDDP resistance in vivo. Results GTSE1 was highly expressed in patients with OS and correlated with poor survival according to the bioinformatics predictions. Elevated GTSE1 expression was detected in OS tissues and cell lines. GTSE1 silencing reduced S/G2 transition and DNA replication, and it increased the CDDP sensitivity and decreased the expression of DNA repair-related biomarkers in MG-63 cells. GTSE1 overexpression in 143B cells led to inverse trends. In vivo, downregulation of GTSE1 strengthened the treating effect of CDDP and significantly repressed growth of xenograft tumors in nude mice. However, overexpression of GTSE1 blocked the anti-tumor effect of CDDP. Conclusion This study demonstrates that GTSE1 is possibly involved in the DNA damage repair and cisplatin resistance in OS.

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