scholarly journals Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts

2005 ◽  
Vol 118 (10) ◽  
pp. 2133-2141 ◽  
Author(s):  
A. Losada
Keyword(s):  
Human Cells ◽  
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 ZW10 links mitotic checkpoint signaling to the structural kinetochore

2005 ◽  
Vol 169 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Geert J.P.L. Kops ◽  
Yumi Kim ◽  
Beth A.A. Weaver ◽  
Yinghui Mao ◽  
Ian McLeod ◽  
...  
Keyword(s):  
Chromosome Instability ◽  
Mitotic Checkpoint ◽  
Human Cells ◽  
Structural Elements ◽  
Checkpoint Signaling ◽  
Daughter Cells ◽  
Primary Mechanism ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80–Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1–Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived “stop anaphase” inhibitor.

scholarly journals Xenopus HJURP and condensin II are required for CENP-A assembly

2011 ◽  
Vol 192 (4) ◽  
pp. 569-582 ◽  
Author(s):  
Rafael Bernad ◽  
Patricia Sánchez ◽  
Teresa Rivera ◽  
Miriam Rodríguez-Corsino ◽  
Ekaterina Boyarchuk ◽  
...  
Keyword(s):  
Protein A ◽  
Human Cells ◽  
Epigenetic Mark ◽  
Temporal Specificity ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Centromeric Protein ◽  
Xenopus Egg Extracts

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction–recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

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

scholarly journals The Prereplication Complex Recruits XEco2 to Chromatin to Promote Cohesin Acetylation in Xenopus Egg Extracts

2012 ◽  
Vol 22 (11) ◽  
pp. 977-988 ◽  
Author(s):  
Torahiko L. Higashi ◽  
Megumi Ikeda ◽  
Hiroshi Tanaka ◽  
Takuro Nakagawa ◽  
Masashige Bando ◽  
...  
Keyword(s):  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts
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