Implication of RPA32 phosphorylation in S-phase checkpoint signalling at replication forks stalled with aphidicolin in Xenopus egg extracts

2012 ◽  
Vol 427 (4) ◽  
pp. 785-789 ◽  
Author(s):  
Bénédicte Recolin ◽  
Domenico Maiorano
Keyword(s):  
S Phase ◽  
Replication Forks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
S Phase Checkpoint

scholarly journals Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

2011 ◽  
Vol 40 (8) ◽  
pp. 3431-3442 ◽  
Author(s):  
Bénédicte Recolin ◽  
Siem Van Der Laan ◽  
Domenico Maiorano
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
S Phase Checkpoint

scholarly journals The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation

2004 ◽  
Vol 165 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Maren Oehlmann ◽  
Alan J. Score ◽  
J. Julian Blow
Keyword(s):  
S Phase ◽  
Multiple Roles ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
High Affinity ◽  
Chromosome Duplication ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers. This process is dependent on Cdc6, which is recruited to unlicensed origins. Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers. This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed. Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA. We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition. These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication.

Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts

2016 ◽  
Vol 470 (2) ◽  
pp. 405-410 ◽  
Author(s):  
Yuta Nakazaki ◽  
Takashi Tsuyama ◽  
Masayuki Seki ◽  
Mikiko Takahashi ◽  
Takemi Enomoto ◽  
...  
Keyword(s):  
Replication Forks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Rad17 Plays a Central Role in Establishment of the Interaction between TopBP1 and the Rad9-Hus1-Rad1 Complex at Stalled Replication Forks

2010 ◽  
Vol 21 (6) ◽  
pp. 926-935 ◽  
Author(s):  
Joon Lee ◽  
William G. Dunphy
Keyword(s):  
Dependent Manner ◽  
Replication Forks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Stalled Replication Forks ◽  
Hydrolysis Of

Rad17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that Rad17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that Rad17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with Rad17 mutants indicate that binding of ATP to Rad17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by Rad17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, Rad17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both Rad17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a Rad17-dependent manner before activation of Chk1.

Overview of the regulation of S-phase in Xenopus egg extracts

Biochimie ◽  
1995 ◽  
Vol 77 (10) ◽  
pp. 808-816 ◽  
Author(s):  
S CHEVALIER
Keyword(s):  
S Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Cdk2 and newly synthesized cdc2 promote initiation of S-phase in Xenopus egg extracts

1995 ◽  
Vol 84 (1-2) ◽  
pp. 106-106
Author(s):  
Stephane Chevalier ◽  
Jean-pierre Tassan ◽  
Rick Cox ◽  
Michel Philippe ◽  
Christopher.c. Ford
Keyword(s):  
S Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks

2013 ◽  
Vol 24 (9) ◽  
pp. 1343-1353 ◽  
Author(s):  
Joon Lee ◽  
William G. Dunphy
Keyword(s):  
Substantial Reduction ◽  
Nuclease Activity ◽  
Replication Forks ◽  
Wild Type ◽  
Mrn Complex ◽  
Egg Extracts ◽  
Unknown Property ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Stalled Replication Forks

The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIβ-binding protein 1 (TopBP1), Rad17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both Rad17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, Rad17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, Rad17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure–function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.

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