USP11 suppresses CHK1 activation by deubiquitinating CLASPIN

2021 ◽  
Author(s):  
Hongchang Zhao ◽  
Zhifeng Wang ◽  
Min Zhu ◽  
Ji Liao ◽  
Xingzhi Xu
Keyword(s):  
Steady State ◽  
Dna Replication ◽  
Genome Stability ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

AbstractCLASPIN is an essential mediator of ATR-dependent CHK1 activation in the DNA replication checkpoint. K6-linked polyubiquitination of CLASPIN promotes its chromatin loading and subsequent CHK1 activation. Here, we found that ubiquitin-specific protease 11 (USP11) deubiquitinates the K6-linkage polyubiquitinated form of CLASPIN. Under steady-state conditions, USP11 interacts with CLASPIN, reducing CLASPIN K6-linked ubiquitination levels. In response to replication stress, USP11 is phosphorylated by ATR and subsequently disassociated from CLASPIN, promoting CLASPIN chromatin loading, CHK1 activation and ultimately genome stability. Taken together, our findings uncover a novel function of USP11 in negatively regulating CHK1 activation by suppressing CLASPIN chromatin loading.

scholarly journals Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

2020 ◽  
Vol 48 (21) ◽  
pp. 12169-12187
Author(s):  
Rose Westhorpe ◽  
Andrea Keszthelyi ◽  
Nicola E Minchell ◽  
David Jones ◽  
Jonathan Baxter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Stress ◽  
Dna Helicase ◽  
The Other ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Binding Partner ◽  
Dna Replication Checkpoint

Abstract The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

scholarly journals The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription

2008 ◽  
Vol 28 (19) ◽  
pp. 5977-5985 ◽  
Author(s):  
Chaitali Dutta ◽  
Prasanta K. Patel ◽  
Adam Rosebrock ◽  
Anna Oliva ◽  
Janet Leatherwood ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Replication ◽  
Budding Yeast ◽  
Replication Stress ◽  
Phosphorylation Sites ◽  
Transcriptional Program ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint ◽  
Regulatory Similarity

ABSTRACT The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

scholarly journals Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae

2006 ◽  
Vol 175 (5) ◽  
pp. 729-741 ◽  
Author(s):  
Jorrit M. Enserink ◽  
Marcus B. Smolka ◽  
Huilin Zhou ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Morphology ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Proteins ◽  
Checkpoint Kinases ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Δ mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

scholarly journals RecQ-like helicases: the DNA replication checkpoint connection

2000 ◽  
Vol 113 (15) ◽  
pp. 2641-2646 ◽  
Author(s):  
C. Frei ◽  
S.M. Gasser
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Normal Cell ◽  
Genome Stability ◽  
Dna Helicase ◽  
S Phase ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint ◽  
Coherent Picture ◽  
Stability Results

The eukaryotic homologues of the Escherichia coli RecQ DNA helicase play conserved roles in the maintenance of genome stability. Results obtained in yeast and mammalian systems are beginning to form a coherent picture about what these helicases do to ensure normal cell division and why humans who lack these enzymes are cancer prone. Recent data suggest that the yeast enzyme Sgs1p, as well as two human homologues, which are encoded by the Bloom's and Werner's syndrome genes, function during DNA replication and possibly in a replication checkpoint specific to S phase.

scholarly journals RecQ DNA Helicase Rqh1 Promotes Rad3ATR Kinase Signaling in the DNA Replication Checkpoint Pathway of Fission Yeast

2020 ◽  
Vol 40 (17) ◽  
Author(s):  
Nafees Ahamad ◽  
Saman Khan ◽  
Yong-jie Xu
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Stress ◽  
Dna Helicase ◽  
Start Codon ◽  
Helicase Domain ◽  
Random Mutation ◽  
Content Type ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint

ABSTRACT Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome, looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported mutant with a C-to-Y change at position 307 encoded by tel2 (tel2-C307Y mutant) (Y.-J. Xu, S. Khan, A. C. Didier, M. Wozniak, et al., Mol Cell Biol 39:e00175-19, 2019, https://doi.org/10.1128/MCB.00175-19), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations, except for a start codon mutation, are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect, and heterologous expression of human RECQ1, BLM, and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved, and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

2021 ◽  
Vol 118 (38) ◽  
pp. e2109334118
Author(s):  
Albert Serra-Cardona ◽  
Chuanhe Yu ◽  
Xinmin Zhang ◽  
Xu Hua ◽  
Yuan Yao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Mutant Cells ◽  
Lagging Strand

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

scholarly journals Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin

2017 ◽  
Vol 114 (19) ◽  
pp. E3766-E3775 ◽  
Author(s):  
Kang Liu ◽  
Fang-Tsyr Lin ◽  
Joshua D. Graves ◽  
Yu-Ju Lee ◽  
Weei-Chin Lin
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
Normal Growth ◽  
Replication Stress ◽  
Dependent Manner ◽  
Gain Of Function ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Activating Function ◽  
Dna Replication Checkpoint

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H–expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: (i) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and (ii) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

scholarly journals The RecQ DNA helicase Rqh1 promotes Rad3ATR kinase signaling in the DNA replication checkpoint pathway of fission yeast

2020 ◽  
Author(s):  
Nafees Ahamad ◽  
Saman Khan ◽  
Yong-jie Xu
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Genome Stability ◽  
Dna Helicase ◽  
Start Codon ◽  
Helicase Domain ◽  
Kinase Signaling ◽  
Random Mutation ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint

ABSTRACTRad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome looking for mutants with defects in Rad3 kinase signaling. In addition to the previously reported tel2-C307Y mutant (1), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations except a start codon mutation are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect and heterologous expression of human RECQ1, BLM and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.

scholarly journals Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

2019 ◽  
Author(s):  
Rose Westhorpe ◽  
Andrea Keszthelyi ◽  
Nicola E. Minchell ◽  
David Jones ◽  
Jonathan Baxter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Stress ◽  
Dna Helicase ◽  
The Other ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Binding Partner ◽  
Dna Replication Checkpoint

AbstractThe highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

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