scholarly journals CtIP interacts with TopBP1 and Nbs1 in the response to double-stranded DNA breaks (DSBs) in Xenopus egg extracts

Cell Cycle ◽  
2011 ◽  
Vol 10 (3) ◽  
pp. 469-480 ◽  
Author(s):  
Juan S. Ramírez-Lugo ◽  
Hae Yong Yoo ◽  
Su Jin Yoon ◽  
William G. Dunphy
Keyword(s):  
Dna Breaks ◽  
Double Stranded Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals The Mre11-Rad50-Nbs1 Complex Mediates Activation of TopBP1 by ATM

2009 ◽  
Vol 20 (9) ◽  
pp. 2351-2360 ◽  
Author(s):  
Hae Yong Yoo ◽  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Human Cells ◽  
Dna Breaks ◽  
Checkpoint Response ◽  
Mrn Complex ◽  
Functional Studies ◽  
Double Stranded Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs.

scholarly journals Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases

2004 ◽  
Vol 165 (6) ◽  
pp. 801-812 ◽  
Author(s):  
Wenhui Li ◽  
Soo-Mi Kim ◽  
Joon Lee ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Chromosomal Abnormalities ◽  
Sister Chromatid Exchanges ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Chromatid Exchanges ◽  
Chromosomal Defects

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIα (Xtop3α) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3α has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

scholarly journals MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication

2017 ◽  
Vol 28 (22) ◽  
pp. 2998-3012 ◽  
Author(s):  
Akiko Kumagai ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
G4 Dna ◽  
G Quadruplex ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication ◽  
Xenopus Egg Extracts

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation.

scholarly journals Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts

2007 ◽  
Vol 1773 (4) ◽  
pp. 556-564 ◽  
Author(s):  
Yuji Kumata ◽  
Shusuke Tada ◽  
Yumie Yamanada ◽  
Takashi Tsuyama ◽  
Takayuki Kobayashi ◽  
...  
Keyword(s):  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Double Strand Dna

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

scholarly journals Crk is required for apoptosis in Xenopus egg extracts

1997 ◽  
Vol 16 (2) ◽  
pp. 230-241 ◽  
Author(s):  
E. K. Evans
Keyword(s):  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts
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