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 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.

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 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.

scholarly journals The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

2009 ◽  
Vol 29 (18) ◽  
pp. 5008-5019 ◽  
Author(s):  
Makiko Komata ◽  
Masashige Bando ◽  
Hiroyuki Araki ◽  
Katsuhiko Shirahige
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Terminal Region ◽  
Methyl Methanesulfonate ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Hydroxyurea Treatment ◽  
Dna Replication Checkpoint ◽  
Direct Binding

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

scholarly journals DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

2004 ◽  
Vol 167 (5) ◽  
pp. 841-849 ◽  
Author(s):  
Ayumi Yamada ◽  
Brad Duffy ◽  
Jennifer A. Perry ◽  
Sally Kornbluth
Keyword(s):  
Dna Replication ◽  
Cell Cycle Control ◽  
Checkpoint Control ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Synthetic Lethal ◽  
Mitotic Entry ◽  
Regulatory Module ◽  
Dna Replication Checkpoint ◽  
Control Modules

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7–Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organsims.

scholarly journals DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

Genetics ◽  
2013 ◽  
Vol 194 (2) ◽  
pp. 389-401 ◽  
Author(s):  
Ying-Chou Chen ◽  
Jessica Kenworthy ◽  
Carrie Gabrielse ◽  
Christine Hänni ◽  
Philip Zegerman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Dna Replication Checkpoint

scholarly journals The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci

2000 ◽  
Vol 14 (1) ◽  
pp. 81-96 ◽  
Author(s):  
Christian Frei ◽  
Susan M. Gasser
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
S Phase ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Replication Fork Progression ◽  
Cycle Arrest ◽  
Dna Replication Checkpoint

We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes). We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication. DNA polε and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed. Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest. The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

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 Increased Genome Instability and Telomere Length in the elg1-Deficient Saccharomyces cerevisiae Mutant Are Regulated by S-Phase Checkpoints

2004 ◽  
Vol 3 (6) ◽  
pp. 1557-1566 ◽  
Author(s):  
Soma Banerjee ◽  
Kyungjae Myung
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Telomere Length ◽  
Genome Instability ◽  
Dna Recombination ◽  
Cell Cycle Checkpoints ◽  
Telomere Maintenance ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint

ABSTRACT Gross chromosomal rearrangements (GCRs) are frequently observed in cancer cells. Abnormalities in different DNA metabolism including DNA replication, cell cycle checkpoints, chromatin remodeling, telomere maintenance, and DNA recombination and repair cause GCRs in Saccharomyces cerevisiae. Recently, we used genome-wide screening to identify several genes the deletion of which increases GCRs in S. cerevisiae. Elg1, which was discovered during this screening, functions in DNA replication by participating in an alternative replication factor complex. Here we further characterize the GCR suppression mechanisms observed in the elg1Δ mutant strain in conjunction with the telomere maintenance role of Elg1. The elg1Δ mutation enhanced spontaneous DNA damage and resulted in GCR formation. However, DNA damage due to inactivation of Elg1 activates the intra-S checkpoints, which suppress further GCR formation. The intra-S checkpoints activated by the elg1Δ mutation also suppress GCR formation in strains defective in the DNA replication checkpoint. Lastly, the elg1Δ mutation increases telomere size independently of other previously known telomere maintenance proteins such as the telomerase inhibitor Pif1 or the telomere size regulator Rif1. The increase in telomere length caused by the elg1Δ mutation was suppressed by a defect in the DNA replication checkpoint, which suggests that DNA replication surveillance by Dpb11-Mec1/Tel1-Dun1 also has an important role in telomere length regulation.

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