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 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 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 Cell Cycle Regulation of the Replication Licensing System: Involvement of a Cdk-dependent Inhibitor

1997 ◽  
Vol 136 (1) ◽  
pp. 125-135 ◽  
Author(s):  
Hiro M. Mahbubani ◽  
James P.J. Chong ◽  
Stephane Chevalier ◽  
Pia Thömmes ◽  
J. Julian Blow
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Kinase Inhibitor ◽  
Cyclin B ◽  
Initiation Factor ◽  
Chromosomal Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Cycle Regulation

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

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