Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest

Capasso, Holly; Palermo, Carmela; Wan, Shan; Rao, Hui Lan; John, Ulrik P.; O’Connell, Matthew J.; Walworth, Nancy C.

doi:10.1242/jcs.00133

Cited by 148 publications

(211 citation statements)

References 57 publications

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“…Cell lysates were prepared and immunoblotted with a phospho-Chk1 S345 specific antibody, to assess activation of Chk1. 28,29,39 Chk1 S345 phosphorylation was readily detectable in response to HU, Gem and Ara-C (Fig. 1A).…”

Section: Resultsmentioning

confidence: 92%

Replication stress activates DNA polymerase alpha-associated Chk1

Taricani¹,

Shanahan²

2009

Cell Cycle

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Section: Resultsmentioning

confidence: 92%

Replication stress activates DNA polymerase alpha-associated Chk1

Taricani¹,

Shanahan²

2009

Cell Cycle

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“…DNA-dependent protein kinase (DNA-PK) is also a transducer kinase that responds less stringently to the presence of DSB DNA damage and is thought to be involved in early response signalling that, in turn, orchestrates DNA damage repair and cell cycle arrests (Lee and Kim, 2002;Gottlieb and Jackson, 1993). Active ATM and ATR trigger downstream effector kinases Chk2 and Chk1 respectively, which are essential to DNA damage checkpoint responses (Chaturvedi et al, 1999;Rhind and Russel, 2000;Capasso et al, 2002). Furthermore, ATM/ATR transducer kinases phosphorylate a vast number of effector proteins, which provoke the DNA damage cellular endpoints.…”

Section: Endpointsmentioning

confidence: 99%

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

Smith¹,

Dejsuphong

Balestrini³

et al. 2009

Nat Cell Biol

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The effects of ATM and ATR signalling induced by chromosomal breakage have been described extensively in modulating cell cycle progression up to the onset of mitosis.However, DNA damage checkpoint responses in mitotic cells are not well understood.This thesis reports on the effects of double strand breaks on the progression of mitosis.We found ATM and ATR activation can occur in mitotic Xenopus laevis egg extract and the induction of ATM and ATR by chromosomal breakages inhibits spindle assembly in both Xenopus egg extract and somatic cells. The delay in mitotic progression induced by ATM and ATR was found not to involve major spindle assembly factors activities such as, Cdk1, Plx1 and RCC1/Ran-GTP. However, normal anastral spindles formation around linear DNA coated beads, which can activate ATM and ATR, linked centrosome-driven spindle assembly to ATM and ATR dependent spindle defects. cDNA expression library screening was undertaken to identify novel ATM and ATR targets in this mitotic checkpoint pathway, through which the novel centrosomal protein XCEP63 was identified as a likely candidate. Data obtained from depletion and reconstitution of XCEP63 in Xenopus egg extract established that normal centrosome-driven spindle assembly requires XCEP63. Moreover, ATM and ATR phosphorylates XCEP63 on serine 560 and promotes delocalisation from the centrosome. ATM and ATR inhibition or addition of non-phosphorylable XCEP63 recombinant protein mutated at serine 560 prevents spindle assembly abnormalities.These findings suggest that ATM and ATR regulate mitotic events by targeting XCEP63 and centrosome-dependent spindle assembly. This pathway may provide support for DNA repair processes or regulate cell survival in the presence of mitotic DNA damage. 3

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“…Cells were grown to mid-log phase and treated with 40 M CPT for 3 h as described previously (Capasso et al, 2002).…”

Section: Camptothecin Treatmentmentioning

confidence: 99%

“…To test whether the dependency of rid mutant viability on chk1 gene expression reflects a dependency on Chk1 kinase activity, we examined the Chk1 kinase by Western immunoblotting of cell extracts isolated from rid mutants following shift to the critical temperature. Chk1 is phosphorylated in response to DNA damage (Capasso et al, 2002), and phosphorylation is dependent on Rad3, a member of the ATM/ATR family of protein kinases, that is believed to directly phosphorylate and activate Chk1. All strains incorporated an HA-tagged copy of Chk1 replacing the endogenous Chk1 so that the Chk1 protein could be easily visualized by Western blot analysis by using HA antibodies.…”

Section: Chk1 Kinase Is Active In Rid Mutants Grown Under Semipermissmentioning

confidence: 99%

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

Yin

Locovei

D’Urso

2008

MBoC

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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. INTRODUCTIONBefore cell division, all eukaryotic cells replicate their DNA through a highly regulated process that involves the binding of several proteins to origin DNA during the G1 phase of the cell cycle (Bell and Dutta, 2002;Diffley and Labib, 2002;Diffley, 2004). The first step in the initiation of DNA replication requires the binding of a six-member hetero-protein complex called Orc to replication origins, marking these sites for subsequent assembly of prereplicative complexes (pre-RCs). The assembly of pre-RCs is believed to occur in early G1, and it requires the loading of Mcm proteins to origin DNA. Two additional proteins Cdc18 and Cdt1 recruit Mcms to DNA and conversion of the pre-RC to an active initiation complex requires activation of both Cdk1 and Hsk1 kinases. Phosphorylation of key components of the pre-RC is believed to result in a reorganization of the origin-associated complex that allows the binding of additional replication proteins, including DNA polymerases and single-strand DNA binding protein. The Mcm complex, with its intrinsic ATPase activity, is believed to participate in the unwinding of DNA during DNA synthesis (Ishimi, 1997;Lee and Hurwitz, 2001).Analysis of cell cycle mutants in both Saccharomyces cerevisiae and Schizosaccharomyces pombe has led to the identification of a number of proteins that are involved in both the initiation and elongation of DNA replication. In addition to the previously mentioned Orc, Cdc18, Cdt1, and Mcm proteins, they include the Gins complex (Alberghina et al., 1983;Takayama et al., 2003;Gambus et al., 2006;Labib and Gambus, 2007), Sna41/Cdc45 (Miyake and Yamashita, 1998;Uchiyama et al., 2001a,b), Sld2, and Sld3 (Kamimura et al., 1998Nakajima an...

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Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest

Cited by 148 publications

References 57 publications

Replication stress activates DNA polymerase alpha-associated Chk1

Replication stress activates DNA polymerase alpha-associated Chk1

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

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

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