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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In response to DNA damage or replication stress, the protein kinase ATR is activated and subsequently transduces genotoxic signals to cell cycle control and DNA repair machinery through phosphorylation of a number of downstream substrates. Very little is known about the molecular mechanism by which ATR is activated in response to genotoxic insults. In this report, we demonstrate that protein phosphatase 5 (PP5) is required for the ATR-mediated checkpoint activation. PP5 forms a complex with ATR in a genotoxic stress-inducible manner. Interference with the expression or the activity of PP5 leads to impairment of the ATR-mediated phosphorylation of hRad17 and Chk1 after UV or hydroxyurea treatment. Similar results are obtained in ATM-deficient cells, suggesting that the observed defect in checkpoint signaling is the consequence of impaired functional interaction between ATR and PP5. In cells exposed to UV irradiation, PP5 is required to elicit an appropriate S-phase checkpoint response. In addition, loss of PP5 leads to premature mitosis after hydroxyurea treatment. Interestingly, reduced PP5 activity exerts differential effects on the formation of intranuclear foci by ATR and replication protein A, implicating a functional role for PP5 in a specific stage of the checkpoint signaling pathway. Taken together, our results suggest that PP5 plays a critical role in the ATR-mediated checkpoint activation.
Mol Cell Biol 2005 Nov
PMID:Protein phosphatase 5 is required for ATR-mediated checkpoint activation. 1626 Jun 6

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.
Mol Biol Cell 2006 Jan
PMID:Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress. 1628 Mar 59

RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.
Mol Cell Biol 2005 Dec
PMID:Inactivation of Ku-mediated end joining suppresses mec1Delta lethality by depleting the ribonucleotide reductase inhibitor Sml1 through a pathway controlled by Tel1 kinase and the Mre11 complex. 1628 75

The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.
Mol Cell Biol 2005 Dec
PMID:Autorepression of rfx1 gene expression: functional conservation from yeast to humans in response to DNA replication arrest. 1628 76

The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as gamma-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that gamma-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in gamma-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.
Mol Cell Biol 2005 Dec
PMID:Cooperative control of Crb2 by ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast. 1631 98

We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.
Mol Cell Biol 2006 Jan
PMID:Recruitment of DNA damage checkpoint proteins to damage in transcribed and nontranscribed sequences. 1635 78

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.
Mol Biol Cell 2006 Apr
PMID:Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus. 1643 14

DNA-damage checkpoints are activated to arrest cells and promote survival upon genotoxic challenge. Efforts have been taken to target checkpoint kinase 1 (chk1; approved gene symbol CHEK1), a crucial checkpoint modulator, for therapeutic intervention. However, improvement of the potency and specificity of such therapeutics remains a major challenge. This prompted us to develop a novel chk1-targeting strategy by constructing a potent oncolytic adenovirus (M2). M2 was generated by combining two artificial features into a wild-type adenovirus type 5 genome. First, M2 was engineered with a 27-bp deletion in the E1A region to confer tumor-selective replication. Second, an antisense chk1 cDNA was substituted for viral E3 6.7K and gp19K genes. In this design, M2 exploited the adenovirus E3 promoters to express antisense chk1 cDNA in a viral replication-dependent fashion and preferentially silenced the chk1 gene in tumor cells. By virtue of combining oncolysis with chk1 targeting, M2 exhibited potent antitumoral efficacy in vitro and in vivo. Systemic administration of M2, plus a low dose of cisplatin, cured 80% of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. These findings have directed us toward the development of novel oncolytic adenoviruses that will be potentially applicable to a wide range of molecular-based therapeutics.
Mol Ther 2006 May
PMID:Selective targeting of checkpoint kinase 1 in tumor cells with a novel potent oncolytic adenovirus. 1645 49

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.
Mol Cell Biol 2006 Mar
PMID:Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint. 1647 16

Pierisin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono-ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N(2) of dG in DNA. Resulting mono-ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis. However, little is known about checkpoint responses elicited in mammalian cells by the formation of such bulky DNA adducts. In the present study, it was shown that DNA polymerases were blocked at the specific site of mono-ADP-ribosylated dG, which might lead to the replication stress. Pierisin-1 treatment of HeLa cells was found to induce an intra-S-phase arrest through both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) and ATM pathways, and ATR pathway also contributes to a G(2)-M-phase delay. In the colony survival assays, Rad17(-/-) DT40 cells showed greater sensitivity to pierisin-1-induced cytotoxicity than wild-type and ATM(-/-) DT40 cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Furthermore, apoptotic 50-kb DNA fragmentation was observed in the HeLa cells, which was well correlated with occurrence of phosphorylation of Chk2. These results thus suggest that pierisin-1 treatment primarily activates ATR pathway and eventually activates ATM pathway as a result of the induction of apoptosis. From these findings, it is suggested that mono-ADP-ribosylation of DNA causes a specific type of fork blockage that induces checkpoint activation and signaling.
Mol Cancer Res 2006 Feb
PMID:Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest evoked by pierisin-1. 1651 43


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