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

2009 ◽  
Vol 20 (3) ◽  
pp. 819-833 ◽  
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.

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

2008 ◽  
Vol 180 (6) ◽  
pp. 1073-1086 ◽  
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.

scholarly journals Critical Role for Mouse Hus1 in an S-Phase DNA Damage Cell Cycle Checkpoint

2003 ◽  
Vol 23 (3) ◽  
pp. 791-803 ◽  
Author(s):  
Robert S. Weiss ◽  
Philip Leder ◽  
Cyrus Vaziri
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Critical Role ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Null Mouse ◽  
Cycle Checkpoint ◽  
S Phase Checkpoint

ABSTRACT Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1− fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G2/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

scholarly journals The Fission Yeast Rad32 (Mre11)-Rad50-Nbs1 Complex Is Required for the S-Phase DNA Damage Checkpoint

2003 ◽  
Vol 23 (18) ◽  
pp. 6564-6573 ◽  
Author(s):  
Charly Chahwan ◽  
Toru M. Nakamura ◽  
Sasirekha Sivakumar ◽  
Paul Russell ◽  
Nicholas Rhind
Keyword(s):  
Dna Damage ◽  
Fission Yeast ◽  
Chromosome Instability ◽  
S Phase ◽  
Comparative Genomic ◽  
Dna Damage Checkpoint ◽  
Checkpoint Signaling ◽  
Damage Repair ◽  
Genomic Approach ◽  
Telomere Regulation

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.

scholarly journals Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
Author(s):  
Ling Yin ◽  
Alexandra Monica Locovei ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Damage Checkpoint ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Fork Stalling ◽  
S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

scholarly journals Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

Oncogene ◽  
2006 ◽  
Vol 25 (44) ◽  
pp. 5921-5932 ◽  
Author(s):  
T Shimura ◽  
M Toyoshima ◽  
S K Adiga ◽  
T Kunoh ◽  
H Nagai ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Low Dose ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Replication Fork Progression

scholarly journals Visualization of Altered Replication Dynamics after DNA Damage in Human Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 20067-20075 ◽  
Author(s):  
Catherine J. Merrick ◽  
Dean Jackson ◽  
John F. X. Diffley
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Replication Fork ◽  
S Phase ◽  
High Rate ◽  
Yeast Cells ◽  
Origin Firing ◽  
Large Numbers ◽  
Dependent Inhibition ◽  
Fork Stalling

Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase.

scholarly journals Checkpoint regulation of replication forks: global or local?

2013 ◽  
Vol 41 (6) ◽  
pp. 1701-1705 ◽  
Author(s):  
Divya Ramalingam Iyer ◽  
Nicholas Rhind
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Dna Damage Checkpoint ◽  
Replication Forks ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Late Origin ◽  
Stalled Replication Forks

Cell-cycle checkpoints are generally global in nature: one unattached kinetochore prevents the segregation of all chromosomes; stalled replication forks inhibit late origin firing throughout the genome. A potential exception to this rule is the regulation of replication fork progression by the S-phase DNA damage checkpoint. In this case, it is possible that the checkpoint is global, and it slows all replication forks in the genome. However, it is also possible that the checkpoint acts locally at sites of DNA damage, and only slows those forks that encounter DNA damage. Whether the checkpoint regulates forks globally or locally has important mechanistic implications for how replication forks deal with damaged DNA during S-phase.

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

2009 ◽  
Vol 20 (6) ◽  
pp. 1683-1694 ◽  
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.

scholarly journals A Ddc2-Rad53 Fusion Protein Can Bypass the Requirements for RAD9 and MRC1 in Rad53 Activation

2004 ◽  
Vol 15 (12) ◽  
pp. 5443-5455 ◽  
Author(s):  
Soo-Jung Lee ◽  
Jimmy K. Duong ◽  
David F. Stern
Keyword(s):  
Dna Damage ◽  
Fusion Proteins ◽  
Essential Role ◽  
Dependent Manner ◽  
Signaling System ◽  
Methyl Methane Sulfonate ◽  
Methane Sulfonate ◽  
Checkpoint Signaling ◽  
Binding Partner ◽  
Mediator Protein

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a “mediator” protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Δrad9 or Δrad9Δmrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Δrad9Δmrc1 cells. In addition, expression of Ddc2-Rad53p in Δrad24Δrad17Δmec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.

Sign in / Sign up

Export Citation Format

Share Document