Cdc2 Tyrosine Phosphorylation is Not Required for the S-Phase DNA Damage Checkpoint in Fission Yeast

Kommajosyula, Naveen; Rhind, Nick

doi:10.4161/cc.5.21.3423

Cited by 15 publications

(15 citation statements)

References 31 publications

(54 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Origin firing is also regulated in fission yeast (Kumar and Huberman 2009). However, Cdc25 regulation is not required in fission yeast to slow replication (Kommajosyula and Rhind 2006). Furthermore, our results show that slowing is abrogated in nbs1D sfr1D double mutants or mus81D single mutants ( Figure 3B and Willis and Rhind 2009).…”

Section: Mrn Mutants Display Partial Defects In Slowingsupporting

confidence: 61%

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

Willis

Rhind

2010

Genetics

View full text Add to dashboard Cite

The Mre11-Rad50-Nbs1 (MRN) heterotrimer plays various and complex roles in DNA damage repair and checkpoint signaling. Its role in activating Ataxia-Telangiectasia Mutated (ATM), the central checkpoint kinase in the metazoan double-strand break response, has been well studied. However, its function in the checkpoint independent of ATM activation, as well as functions that are completely checkpoint independent, are less well understood. In fission yeast, DNA damage checkpoint signaling requires Rad3, the homolog of the ATR (ATM and Rad3-related) kinase, not Tel1, the ATM homolog, allowing us to dissect MRN's ATM-independent S-phase DNA damage checkpoint roles from its role in ATM activation. We find that MRN is involved in Rad3 (ATR)-dependent checkpoint signaling in S phase, but not G2, suggesting that MRN is involved in ATR activation through its role in replication fork metabolism. In addition, we define a role for MRN in the S-phase DNA damage checkpoint-dependent slowing of replication that is independent of its role in checkpoint signaling. Genetic interactions between MRN and Rhp51, the fission yeast Rad51 homolog, lead us to suggest that MRN participates in checkpoint-dependent replication slowing through negative regulation of recombination.

show abstract

Section: Mrn Mutants Display Partial Defects In Slowingsupporting

confidence: 61%

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

Willis

Rhind

2010

Genetics

View full text Add to dashboard Cite

show abstract

“…These results allow us to conclude that deletion of flp1 does not significantly affect Cdc25p protein levels or Cdc2p Y15 phosphorylation in response to replication stress caused by nucleotide depletion. Even though in fission yeast the strict requirement of Cdc2 Y15 phosphorylation as the key mechanism for Cdk/cyclin inhibition in response to S phase DNA damage is still in dispute Rhind and Russell, 1998b;Kommajosyula and Rhind, 2006), it is, indeed, a fact that p34 cdc2 becomes hyperphosphorylated on tyrosine 15 upon HU, MMS, or UV light treatments (O'Connell et al, 1997;Chu et al, 2007). In this context, it is surprising to see that Flp1p, the phosphatase that reverts Cdk phosphorylation events on Cdc25p down-regulating its activity as cells exit from mitosis, does not seem to exert such control under replicative stress.…”

mentioning

confidence: 99%

Cds1 Controls the Release of Cdc14-like Phosphatase Flp1 from the Nucleolus to Drive Full Activation of the Checkpoint Response to Replication Stress in Fission Yeast

Díaz-Cuervo

Bueno

2008

MBoC

View full text Add to dashboard Cite

The Cdc14p-like phosphatase Flp1p (also known as Clp1p) is regulated by cell cycle-dependent changes in its subcellular localization. Flp1p is restricted to the nucleolus and spindle pole body until prophase, when it is dispersed throughout the nucleus, mitotic spindle, and medial ring. Once released, Flp1p antagonizes Cdc2p/cyclin activity by reverting Cdc2p-phosphorylation sites on Cdc25p. On replication stress, ataxia-telangiectasia mutated/ATM/Rad3-related kinase Rad3p activates Cds1p, which phosphorylates key proteins ensuring the stability of stalled DNA replication forks. Here, we show that replication stress induces changes in the subcellular localization of Flp1p in a checkpoint-dependent manner. Active Cds1p checkpoint kinase is required to release Flp1p into the nucleus. Consistently, a Flp1p mutant (flp1-9A) lacking all potential Cds1p phosphorylation sites fails to relocate in response to replication blocks and, similarly to cells lacking flp1 (⌬flp1), presents defects in checkpoint response to replication stress. ⌬flp1 cells accumulate reduced levels of a less active Cds1p kinase in hydroxyurea (HU), indicating that nuclear Flp1p regulates Cds1p full activation. Consistently, ⌬flp1 and flp1-9A have an increased percentage of Rad22p-recombination foci during HU treatment. Together, our data show that by releasing Flp1p into the nucleus Cds1p checkpoint kinase modulates its own full activation during replication stress. INTRODUCTIONAdequate genome replication and transfer to daughter cells is compromised in the course of each cell cycle by both intraand extracellular factors that perturb the stability of the genome. Molecular mechanisms to ensure genome integrity and to cope with DNA harassment were developed in eukaryotes likely at early stages of evolution, and they are consequently conserved. Therefore, a replication checkpoint ensures genomic integrity and cell survival when cells are not able to correctly replicate their genetic material, due to limitation in nucleotide pools or to deleterious alterations in template DNA.Serine/threonine phosphatase Flp1p (cdc fourteen-like phosphatase, also called Clp1p; hereafter referred as Flp1p) has been proved to control rapid degradation of Cdc25p at the end of mitosis (Esteban et al., 2004;Wolfe and Gould, 2004), which results in enhanced inhibitory Y15 phosphorylation of Cdc2p and in the corresponding loss of kinase activity necessary for mitotic exit. Flp1p is required for the ubiquitination of Cdc25p by the anaphase promoting complex/cyclosome at this cell cycle stage, and cells deleted for flp1 ϩ present higher basal levels of Cdc25p. Interestingly, this role may be conserved in higher eukaryotes as hCdc14Ap is involved in the cell cycle regulation of hCdc25Ap stability through dephosphorylation of Serines 115 and 329 of the mitotic inducer in human cells (Esteban et al., 2006). In fission yeast, ⌬flp1 cells enter mitosis at a reduced cell size, presenting a wee phenotype (Cueille et al., 2001;Trautmann et al., 2001). Overexpression of Flp1p arrests ...

show abstract

“…We concluded that, in fission yeast, the Rad33Cds1ٜCdc253Cdc2 pathway forms a checkpoint signaling module very similar to the corresponding one of vertebrates. However, Kommajosyula and Rhind were not able to repeat our observations regarding the roles of Cdc25 and Cdc2 (22), so the relevance of Cdc25 and Cdc2 to checkpoint-induced slowing of S phase in fission yeast has remained uncertain until now. In addition, whether S phase in MMS-treated fission yeast cells is slowed by inhibition of origin firing, by reduction in rate of fork movement, or by a combination of these has been equally unclear.…”

mentioning

confidence: 55%

“…However, some of their experiments were carried out at 0.03% MMS, which is twice as high as the highest concentration employed by us (0.015%). Furthermore, the effective concentration of the batch of MMS employed by Kommajosyula and Rhind (22) may have been greater than the effective concentration of the batch employed by us, for the following reasons. First, we have observed considerable batch-tobatch variation in effective concentrations of MMS, as judged by the concentration required to produce a given biological effect (which, in our case, is the retardation of progression through S phase in wild-type cells).…”

Section: Discussionmentioning

confidence: 96%

See 1 more Smart Citation

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

Kumar

Huberman

2009

Molecular and Cellular Biology

View full text Add to dashboard Cite

To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G 1 ) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.In response to low levels of the DNA-alkylating agent methyl methanesulfonate (MMS), wild-type yeast cells slow their progression through S phase, while cells lacking the appropriate upstream checkpoint kinase (Mec1 in the budding yeast Saccharomyces cerevisiae; Rad3 in the fission yeast Schizosaccharomyces pombe) or the appropriate downstream checkpoint kinase (Rad53 in budding yeast, Cds1 in fission yeast) fail to do so. Other DNA-damaging agents also cause a checkpoint-dependent slowing of S phase, in vertebrates as well as in yeasts. This slowing of S phase in response to DNA damage is sometimes called the "intra-S-phase" checkpoint (3,6,22,23,26,28,36,37,45,53). Here we shall refer to it as the "S-phase damage" checkpoint.Prior to this report, the downstream portions of the checkpoint pathway(s) that slow S phase in response to DNA damage in fission yeast were unclear. However, the upstream portions of these pathways in fission yeast and other organisms have been partially elucidated, and downstream mechanisms in other organisms have been partially clarified. In all studied systems, upon detection of DNA damage in S phase, checkpoint proteins initiate a phosphorylation cascade that ultimately leads to slowing of replication. Upstream signaling in these systems involves the activation of one or more of the phosphatidylinositol-3-kinase-like protein kinases (PIK kinases; ATR and/or ATM in humans, Mec1 and/or Tel1 in budding yeast, and Rad3 in fission yeast). The activated PIK kinases then phosphorylate several proteins, including certain Ser/Thr kinases (Chk1 and/or Chk2 in humans, Rad53 in budding yeast, and Cds1 in fission yeast). These kinases, in turn, phosphorylate other substrates that, directly or indirectly, mediate the slowing of S phase (reviewed in reference 3).In budding yeast, two different mechanisms were shown to slow S phase upon DNA damage by MMS. Of these, one mechanism, inhibition of late-firing origins, depended on the Mec1-Rad53 checkpoint pathway (45, 53), while the other mechanism, inhibition of replication fo...

show abstract

Cdc2 Tyrosine Phosphorylation is Not Required for the S-Phase DNA Damage Checkpoint in Fission Yeast

Cited by 15 publications

References 31 publications

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

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

Cds1 Controls the Release of Cdc14-like Phosphatase Flp1 from the Nucleolus to Drive Full Activation of the Checkpoint Response to Replication Stress in Fission Yeast

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

Contact Info

Product

Resources

About