scholarly journals XRad17 Is Required for the Activation of XChk1 But Not XCds1 during Checkpoint Signaling in Xenopus

2003 ◽  
Vol 14 (9) ◽  
pp. 3898-3910 ◽  
Author(s):  
Rhiannon E. Jones ◽  
J. Ross Chapman ◽  
Chandrakala Puligilla ◽  
Johanne M. Murray ◽  
Antony M. Car ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Replication Block ◽  
Processivity Factor ◽  
Factor C ◽  
Stalled Replication Forks

The DNA damage/replication checkpoints act by sensing the presence of damaged DNA or stalled replication forks and initiate signaling pathways that arrest cell cycle progression. Here we report the cloning and characterization of Xenopus orthologues of the RFCand PCNA-related checkpoint proteins. XRad17 shares regions of homology with the five subunits of Replication factor C. XRad9, XRad1, and XHus1 (components of the 9-1-1 complex) all show homology to the DNA polymerase processivity factor PCNA. We demonstrate that these proteins associate with chromatin and are phosphorylated when replication is inhibited by aphidicolin. Phosphorylation of X9-1-1 is caffeine sensitive, but the chromatin association of XRad17 and the X9-1-1 complex after replication block is unaffected by caffeine. This suggests that the X9-1-1 complex can associate with chromatin independently of XAtm/XAtr activity. We further demonstrate that XRad17 is essential for the chromatin binding and checkpoint-dependent phosphorylation of X9-1-1 and for the activation of XChk1 when the replication checkpoint is induced by aphidicolin. XRad17 is not, however, required for the activation of XCds1 in response to dsDNA ends.

scholarly journals Spindle Checkpoint Signaling Requires the Mis6 Kinetochore Subcomplex, Which Interacts with Mad2 and Mitotic Spindles

2005 ◽  
Vol 16 (8) ◽  
pp. 3666-3677 ◽  
Author(s):  
Shigeaki Saitoh ◽  
Kojiro Ishii ◽  
Yasuyo Kobayashi ◽  
Kohta Takahashi
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Cycle Progression ◽  
Centromeric Chromatin ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Potential Binding ◽  
Evolutionarily Conserved

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle–kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.

scholarly journals Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

2006 ◽  
Vol 173 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Shan Yan ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Sperm Chromatin ◽  
Checkpoint Control ◽  
Direct Role ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Oligonucleotide Duplex ◽  
Stalled Replication Forks

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3–related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

A method for assaying the sensitivity of Drosophila replication checkpoint mutants to anti-cancer and DNA-damaging drugs.

Genome ◽  
2002 ◽  
Vol 45 (5) ◽  
pp. 881-889 ◽  
Author(s):  
Colleen M Radcliffe ◽  
Elizabeth A Silva ◽  
Shelagh D Campbell
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Tumor Formation ◽  
Cycle Progression ◽  
Replication Checkpoint ◽  
Anti Cancer ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes ◽  
Regulate Cell Cycle Progression

In multi-cellular organisms, failure to properly regulate cell-cycle progression can result in inappropriate cell death or uncontrolled cell division leading to tumor formation. To guard against such events, conserved regulatory mechanisms called "checkpoints" block progression into mitosis in response to DNA damage and incomplete replication, as well as in response to other signals. Checkpoint mutants in organisms as diverse as yeast and humans are sensitive to various chemical agents that inhibit DNA replication or cause DNA damage. This phenomenon is the primary rationale for chemotherapy, which uses drugs that preferentially target tumor cells with compromised checkpoints. In this study, we demonstrate the use of Drosophila checkpoint mutants as a system for assaying the effects of various DNA-damaging and anti-cancer agents in a developing multicellular organism. Dwee1, grp and mei-41 are genes that encode kinases that function in the DNA replication checkpoint. We tested zygotic mutants of each gene for sensitivity to the DNA replication inhibitor hydroxyurea (HU), methyl methanosulfonate (MMS), ara-C, cisplatin, and the oxygen radical generating compound paraquat. The mutants show distinct differences in their sensitivity to each of the drugs tested, suggesting an underlying complexity in the responses of individual checkpoint genes to genotoxic stress.Key words: hydroxyurea (HU), ara-C, cisplatin, methyl methane sulfonate (MMS), paraquat.

Cloning and characterization of a novel intracellular protein p48.2 that negatively regulates cell cycle progression

2009 ◽  
Vol 41 (11) ◽  
pp. 2240-2250 ◽  
Author(s):  
Fan Yang ◽  
Yu-Ping Xu ◽  
Jian Li ◽  
Su-Su Duan ◽  
Ying-Jie Fu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Intracellular Protein ◽  
Cycle Progression

scholarly journals A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase

2002 ◽  
Vol 22 (18) ◽  
pp. 6509-6520 ◽  
Author(s):  
Tetsuo Maruyama ◽  
Andrea Farina ◽  
Anup Dey ◽  
JaeHun Cheong ◽  
Vladimir P. Bermudez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Replication Factor C ◽  
Cycle Progression ◽  
Vitro Analysis ◽  
Factor C ◽  
In Vitro Analysis

ABSTRACT Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G1 upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G1 to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.

Abstract LB-159: Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter

2014 ◽  
Author(s):  
Gabriel Pineda ◽  
Florence Lambert-Fliszar ◽  
Gennarina L. Riso ◽  
Kathleen M. Kane ◽  
Catriona Jamieson
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cycle Progression
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