RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast

Gardner, Richard D.; Putnam, Charles W.; Weinert, Ted

doi:10.1093/emboj/18.11.3173

Cited by 152 publications

(151 citation statements)

References 68 publications

(119 reference statements)

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“…This result extends the previous observation that dun1⌬ is synthetically lethal with rad53-11 (22). In addition, dun1⌬ strains exhibit an increased rate of petite formation, and dun1⌬ mec1-3 or -8 strains exhibit even greater levels of petite formation.…”

Section: Discussionsupporting

confidence: 88%

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1

Zhao

Rothstein²

2002

Proc. Natl. Acad. Sci. U.S.A.

253

265

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Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals. One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis. In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1͞Rad53 (yeast) and ATM͞CHK2 (human) checkpoint pathways. In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1. After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein. This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear. Here we show that Dun1, a downstream kinase of Mec1͞Rad53, genetically and physically interacts with Sml1 in vivo. The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage. As a result, dun1⌬ strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents. Moreover, phospho-Sml1 is absent or dramatically reduced in dun1⌬ cells. Finally, Dun1 can phosphorylate Sml1 in vitro. These results suggest that Dun1 kinase function is the last step required in the Mec1͞Rad53 cascade to remove Sml1 during S phase and after DNA damage. R ibonucleotide reductase (RNR) catalyzes the conversion of NDP to dNDP, the rate-limiting step of dNTP synthesis. RNR activity directly affects the levels and balances of the dNTP pools and subsequently both genetic fidelity and cell viability (1).As an important enzyme in the early stages of DNA synthesis, RNR activity is tightly regulated during DNA replication and repair. Besides its intrinsic allosteric regulation, RNR activity is regulated at the transcriptional level (2) and also by its inhibitor, Sml1 (3, 4). In yeast, these last two types of control both require the evolutionarily conserved checkpoint kinases, Mec1 and Rad53 (5).In response to DNA damage, Mec1 and Rad53 activate the downstream checkpoint kinase Dun1, which leads to the transcriptional induction of the RNR genes (6-8). Such induction is achieved by the relief of transcriptional repression by the Crt1 protein (9). However, the direct downstream substrate(s) of the Dun1 kinase remain unknown. Similar transcriptional induction of the RNR genes also exists in mammals. For example, a human RNR small subunit (p53R2) is induced by p53 after DNA damage, and this induction is crucial for DNA repair (10). Given that the p53 is activated after DNA damage by the Mec1 and Rad53 homologs, ATM͞ATR and CHK2 (11), it is likely that the RNR transcriptional induction circuitry is largely conserved between yeast and mammals.In addition to transcriptional induction of the RNR genes, the Mec1͞Rad53 pathway also increases RNR activity by regulating the removal of the RNR inhibitor, Sml1 (3, 4). Sml1 is a 104-residue peptide that binds t...

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Section: Discussionsupporting

confidence: 88%

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1

Zhao

Rothstein²

2002

Proc. Natl. Acad. Sci. U.S.A.

253

265

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“…6). This conclusion is consistent with the partial cell cycle arrest observed in chk1⌬ and rad53⌬ mutant strains in response to DNA damage (8,9). Rad53 is likely to have additional targets besides Pds1, because 2 K. Ross and O. Cohen-Fix, unpublished observations.…”

Section: Discussionsupporting

confidence: 80%

“…Chk1 and Rad53 are on two parallel branches of the DNA damage checkpoint pathway, and in budding yeast both are required for a complete metaphase cell cycle arrest ( Fig. 1) (8,9). The downstream target of Rad53 that is needed for the mitotic arrest is unknown.…”

mentioning

confidence: 99%

Two Distinct Pathways for Inhibiting Pds1 Ubiquitination in Response to DNA Damage

Agarwal

Tang

et al. 2003

Journal of Biological Chemistry

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The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/C Cdc20 ). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/ C Cdc20 -dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.

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“…A dun1 mutation would be expected to inactivate the checkpoint downstream from assembly of Ddc2.GFP foci, allowing us to monitor checkpoint activation in the absence of checkpoint execution (31,34,35). We found a significant increase in Ddc2.GFP foci in asf1 dun1 mutants, with 45% of cells containing one or more Ddc2.GFP focus (Fig.…”

Section: Analysis Of Spontaneous Checkpoint Protein Assembly In Asf1 mentioning

confidence: 75%

Checkpoint functions are required for normal S-phase progression in Saccharomyces cerevisiae RCAF- and CAF-I-defective mutants

Kats

Albuquerque

Zhou

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The chromatin-assembly factor I (CAF-I) and the replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and play a role in the maintenance of genome stability. Here, we have investigated their role in checkpoints and S-phase progression. FACS analysis of mutants lacking Asf1 or Cac1 as well as various checkpoint proteins indicated that normal rates of S-phase progression in asf1 mutants have a strong requirement for replication checkpoint proteins, whereas normal S-phase progression in cac1 mutants has only a weak requirement for either replication or DNA-damage checkpoint proteins. Furthermore, asf1 mutants had high levels of Ddc2.GFP foci that were further increased in asf1 dun1 double mutants consistent with a requirement for checkpoint proteins in S-phase progression in asf1 mutants, whereas cac1 mutants had much lower levels of Ddc2.GFP foci that were not increased by a dun1 mutation. Our data suggest that RCAF defects lead to unstable replication forks that are then stabilized by replication checkpoint proteins, whereas CAF-I defects likely cause different types of DNA damage.chromatin assembly ͉ DNA-damage response ͉ DNA replication ͉ genome instability

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RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast

Cited by 152 publications

References 68 publications

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1

Two Distinct Pathways for Inhibiting Pds1 Ubiquitination in Response to DNA Damage

Checkpoint functions are required for normal S-phase progression in Saccharomyces cerevisiae RCAF- and CAF-I-defective mutants

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