Involvement of annexin II in DNA replication: evidence from cell-free extracts of Xenopus eggs

1993 ◽  
Vol 105 (2) ◽  
pp. 533-540 ◽  
Author(s):  
J.K. Vishwanatha ◽  
S. Kumble
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Plasmid Dna ◽  
Annexin Ii ◽  
Chromosomal Dna ◽  
Stringent Requirement ◽  
Exogenous Dna ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Higher Eukaryotes

Cell-free extracts of Xenopus eggs efficiently initiate and complete semiconservative DNA replication of exogenously added plasmid DNA. DNA replication in such extracts can be neutralized by a monoclonal antibody (D1/274.5) against human annexin II. Specific immunodepletion of Xenopus annexin II from the egg extracts results in loss of DNA replicative ability. Immunodepletion of annexin II does not prevent nuclear assembly, a stringent requirement for DNA synthesis on exogenous DNA in this system. Replicative ability can be restored to the immunodepleted extracts by the addition of purified human annexin II. These results demonstrate that annexin II is involved in chromosomal DNA replication and has a role in the cell cycle of higher eukaryotes.

scholarly journals Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression.

1994 ◽  
Vol 125 (4) ◽  
pp. 705-719 ◽  
Author(s):  
S Kornbluth ◽  
M Dasso ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Structure ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
A Cell ◽  
Xenopus Egg Extracts

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP-bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

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 An Essential and Cell-Cycle-Dependent ORC Dimerization Cycle Regulates Eukaryotic Chromosomal DNA Replication

Cell Reports ◽  
2020 ◽  
Vol 30 (10) ◽  
pp. 3323-3338.e6
Author(s):  
Aftab Amin ◽  
Rentian Wu ◽  
Man Hei Cheung ◽  
John F. Scott ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Chromosomal Dna ◽  
Cycle Dependent

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 Response of Xenopus Cds1 in Cell-free Extracts to DNA Templates with Double-stranded Ends

2000 ◽  
Vol 11 (5) ◽  
pp. 1535-1546 ◽  
Author(s):  
Zijian Guo ◽  
William G. Dunphy
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Molecules ◽  
Dna Templates ◽  
Cell Cycle Delay ◽  
Checkpoint Kinases ◽  
Double Stranded Dna ◽  
Egg Extracts ◽  
Dna Ends ◽  
Damaged Dna

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that XenopusCds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-freeXenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast,Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.

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 Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport

1997 ◽  
Vol 8 (10) ◽  
pp. 1955-1970 ◽  
Author(s):  
Robert T. Pu ◽  
Mary Dasso
Keyword(s):  
Dna Replication ◽  
Protein Import ◽  
Nuclear Transport ◽  
Small Gtpase ◽  
Stable Complex ◽  
Guanine Nucleotide ◽  
Vitro Assay ◽  
Nuclear Assembly ◽  
Egg Extracts

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 fromXenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.

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