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

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.

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Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.86 ◽

1996 ◽

Vol 16 (1) ◽

pp. 86-93 ◽

Cited By ~ 45

Author(s):

R Kovelman ◽

P Russell

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Replication Checkpoint ◽

Activation Assay ◽

M Phase ◽

Dna Replication Checkpoint ◽

High Level ◽

Tyrosyl Phosphorylation ◽

In Cells

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

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Cdc18p can block mitosis by two independent mechanisms

Journal of Cell Science ◽

10.1242/jcs.111.20.3101 ◽

1998 ◽

Vol 111 (20) ◽

pp. 3101-3108 ◽

Cited By ~ 2

Author(s):

E. Greenwood ◽

H. Nishitani ◽

P. Nurse

Keyword(s):

Dna Replication ◽

S Phase ◽

Checkpoint Control ◽

Replication Checkpoint ◽

Mitotic Kinase ◽

C Terminus ◽

N Terminus ◽

Dna Replication Checkpoint ◽

Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

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DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

The Journal of Cell Biology ◽

10.1083/jcb.200406048 ◽

2004 ◽

Vol 167 (5) ◽

pp. 841-849 ◽

Cited By ~ 15

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.

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Tyrosine Phosphorylation of Cdc2 Is Required for the Replication Checkpoint in Schizosaccharomyces pombe

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.3782 ◽

1998 ◽

Vol 18 (7) ◽

pp. 3782-3787 ◽

Cited By ~ 68

Author(s):

Nicholas Rhind ◽

Paul Russell

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Biochemical Study ◽

Replication Checkpoint ◽

Hydroxyurea Treatment ◽

Activated State ◽

Dna Replication Checkpoint ◽

Cdc2 Activity ◽

In Cells

ABSTRACT The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea. In the fission yeastSchizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism. We have undertaken biochemical and genetic studies to resolve this controversy. We report that the DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis. These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint.

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Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e11-10-0852 ◽

2012 ◽

Vol 23 (6) ◽

pp. 1058-1067 ◽

Cited By ~ 16

Author(s):

Theresa J. Berens ◽

David P. Toczyski

Keyword(s):

Dna Replication ◽

Double Strand Breaks ◽

Sensor Kinase ◽

Replication Forks ◽

Checkpoint Activation ◽

Replication Checkpoint ◽

Strand Breaks ◽

Crucial Step ◽

Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa963 ◽

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.

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Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.672510 ◽

2021 ◽

Vol 9 ◽

Author(s):

Maksym Shyian ◽

David Shore

Keyword(s):

Dna Replication ◽

Nuclear Dna ◽

Protein Complexes ◽

Genome Integrity ◽

Positive Role ◽

Replication Checkpoint ◽

Dna Replication Checkpoint ◽

Protein Barrier

During nuclear DNA replication multiprotein replisome machines have to jointly traverse and duplicate the total length of each chromosome during each cell cycle. At certain genomic locations replisomes encounter tight DNA-protein complexes and slow down. This fork pausing is an active process involving recognition of a protein barrier by the approaching replisome via an evolutionarily conserved Fork Pausing/Protection Complex (FPC). Action of the FPC protects forks from collapse at both programmed and accidental protein barriers, thus promoting genome integrity. In addition, FPC stimulates the DNA replication checkpoint and regulates topological transitions near the replication fork. Eukaryotic cells have been proposed to employ physiological programmed fork pausing for various purposes, such as maintaining copy number at repetitive loci, precluding replication-transcription encounters, regulating kinetochore assembly, or controlling gene conversion events during mating-type switching. Here we review the growing number of approaches used to study replication pausing in vivo and in vitro as well as the characterization of additional factors recently reported to modulate fork pausing in different systems. Specifically, we focus on the positive role of topoisomerases in fork pausing. We describe a model where replisome progression is inherently cautious, which ensures general preservation of fork stability and genome integrity but can also carry out specialized functions at certain loci. Furthermore, we highlight classical and novel outstanding questions in the field and propose venues for addressing them. Given how little is known about replisome pausing at protein barriers in human cells more studies are required to address how conserved these mechanisms are.

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

10.1101/2019.12.17.877811 ◽

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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