The Fission Yeast Rad32(Mre11)–Rad50–Nbs1 Complex Acts Both Upstream and Downstream of Checkpoint Signaling in the S-Phase DNA Damage Checkpoint

ABSTRACT Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G2 DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.

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Esc2 and Sgs1 Act in Functionally Distinct Branches of the Homologous Recombination Repair Pathway inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0877 ◽

2009 ◽

Vol 20 (6) ◽

pp. 1683-1694 ◽

Cited By ~ 41

Author(s):

Hocine W. Mankouri ◽

Hien-Ping Ngo ◽

Ian D. Hickson

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Structural Similarity ◽

S Phase ◽

Homologous Recombination Repair ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Checkpoint Response ◽

Recombination Repair ◽

Anemia Group

Esc2 is a member of the RENi family of SUMO-like domain proteins and is implicated in gene silencing in Saccharomyces cerevisiae. Here, we identify a dual role for Esc2 during S-phase in mediating both intra-S-phase DNA damage checkpoint signaling and preventing the accumulation of Rad51-dependent homologous recombination repair (HRR) intermediates. These roles are qualitatively similar to those of Sgs1, the yeast ortholog of the human Bloom's syndrome protein, BLM. However, whereas mutation of either ESC2 or SGS1 leads to the accumulation of unprocessed HRR intermediates in the presence of MMS, the accumulation of these structures in esc2 (but not sgs1) mutants is entirely dependent on Mph1, a protein that shows structural similarity to the Fanconi anemia group M protein (FANCM). In the absence of both Esc2 and Sgs1, the intra-S-phase DNA damage checkpoint response is compromised after exposure to MMS, and sgs1esc2 cells attempt to undergo mitosis with unprocessed HRR intermediates. We propose a model whereby Esc2 acts in an Mph1-dependent process, separately from Sgs1, to influence the repair/tolerance of MMS-induced lesions during S-phase.

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Mus81, Rhp51(Rad51), and Rqh1 Form an Epistatic Pathway Required for the S-Phase DNA Damage Checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0798 ◽

2009 ◽

Vol 20 (3) ◽

pp. 819-833 ◽

Cited By ~ 27

Author(s):

Nicholas Willis ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Replication Fork ◽

Alkylating Agent ◽

S Phase ◽

Negative Regulation ◽

Dna Damage Checkpoint ◽

Methyl Methane Sulfonate ◽

Methane Sulfonate ◽

Checkpoint Signaling

The S-phase DNA damage checkpoint slows the rate of DNA synthesis in response to damage during replication. In the fission yeast Schizosaccharomyces pombe, Cds1, the S-phase-specific checkpoint effector kinase, is required for checkpoint signaling and replication slowing; upon treatment with the alkylating agent methyl methane sulfonate, cds1Δ mutants display a complete checkpoint defect. We have identified proteins downstream of Cds1 required for checkpoint-dependant slowing, including the structure-specific endonuclease Mus81 and the helicase Rqh1, which are implicated in replication fork stability and the negative regulation of recombination. Removing Rhp51, the Rad51 recombinase homologue, suppresses the slowing defect of rqh1Δ mutants, but not that of mus81Δ mutant, defining an epistatic pathway in which mus81 is epistatic to rhp51 and rhp51 is epistatic to rqh1. We propose that restraining recombination is required for the slowing of replication in response to DNA damage.

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Mechanism of Caffeine-Induced Checkpoint Override in Fission Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.12.4288-4294.2000 ◽

2000 ◽

Vol 20 (12) ◽

pp. 4288-4294 ◽

Cited By ~ 58

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.

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Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins

The Journal of Cell Biology ◽

10.1083/jcb.200706009 ◽

2008 ◽

Vol 180 (6) ◽

pp. 1073-1086 ◽

Cited By ~ 8

Author(s):

Julie M. Caldwell ◽

Yinhuai Chen ◽

Kaila L. Schollaert ◽

James F. Theis ◽

George F. Babcock ◽

...

Keyword(s):

Dna Damage ◽

Replication Fork ◽

S Phase ◽

Dna Damage Checkpoint ◽

Replication Forks ◽

Checkpoint Signaling ◽

Checkpoint Pathway ◽

Origin Licensing ◽

Pause Site ◽

S Phase Checkpoint

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Δ cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Δ dun1Δ cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin–Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.

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The Role of MRN in the S-Phase DNA Damage Checkpoint Is Independent of Its Ctp1-dependent Roles in Double-Strand Break Repair and Checkpoint Signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e08-09-0986 ◽

2009 ◽

Vol 20 (7) ◽

pp. 2096-2107 ◽

Cited By ~ 10

Author(s):

Mary E. Porter-Goff ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Double Strand Break Repair ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Break Repair ◽

S Phase Checkpoint

The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5′ resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure–function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with ctp1Δ, are defective in double-strand break repair and most other functions of the complex, but they maintain an intact S phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads us to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.

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