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 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 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.

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.

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