scholarly journals DNA replication of mitotic chromatin in Xenopus egg extracts

2003 ◽  
Vol 100 (23) ◽  
pp. 13241-13246 ◽  
Author(s):  
T. A. Prokhorova ◽  
K. Mowrer ◽  
C. H. Gilbert ◽  
J. C. Walter
Keyword(s):  
Dna Replication ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs

1991 ◽  
Vol 98 (3) ◽  
pp. 271-279
Author(s):  
J. Meier ◽  
K.H. Campbell ◽  
C.C. Ford ◽  
R. Stick ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Membrane Structures ◽  
Chromatin Decondensation ◽  
Immunoblotting Analysis ◽  
Nuclear Assembly ◽  
Contrast Microscopy ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xenopus egg extracts, which support nuclear assembly and DNA replication, were functionally depleted of lamin LIII by inoculating them with monoclonal anti-lamin antibodies. Phase-contrast microscopy and electron-microscopy studies indicated that lamin-depleted extracts supported efficient chromatin decondensation, and assembly of double membrane structures and nuclear pores on demembranated sperm heads. Immunofluorescence microscopy suggests that lamin-antibody complexes are transported across the nuclear membrane but do not assemble into a lamina. These findings were confirmed by immunoblotting analysis of isolated nuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking a lamina were unable to initiate DNA replication and that, although such nuclei could import proteins required for DNA replication (e.g. PCNA), these proteins were apparently not organized into replicon clusters.

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 Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization

1997 ◽  
Vol 138 (3) ◽  
pp. 615-628 ◽  
Author(s):  
Rebecca Heald ◽  
Régis Tournebize ◽  
Anja Habermann ◽  
Eric Karsenti ◽  
Anthony Hyman
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Xenopus Egg Extracts ◽  
Microtubule Stabilizing Agent ◽  
Mitotic Chromatin

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

scholarly journals Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts

2013 ◽  
Vol 41 (16) ◽  
pp. 7725-7737 ◽  
Author(s):  
Elaine M. Taylor ◽  
Nicola M. Bonsu ◽  
R. Jordan Price ◽  
Howard D. Lindsay
Keyword(s):  
Dna Replication ◽  
Chromosomal Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

2021 ◽  
Author(s):  
Tamar Kavlashvili ◽  
James M Dewar
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Replicative Helicase ◽  
Before And After ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Biochemical Approach ◽  
Key Steps ◽  
Xenopus Egg Extracts

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

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