scholarly journals The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR

1995 ◽  
Vol 108 (1) ◽  
pp. 235-244 ◽  
Author(s):  
J. Murphy ◽  
C.M. Crompton ◽  
S. Hainey ◽  
G.A. Codd ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Nuclear Lamina ◽  
Nuclear Antigen ◽  
Cyanobacterial Toxin ◽  
Nuclear Assembly ◽  
Unusual Distribution ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly

Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supplemented with the protein phosphatase inhibitor microcystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.

Nuclei that lack a lamina accumulate karyophilic proteins and assemble a nuclear matrix

1993 ◽  
Vol 106 (1) ◽  
pp. 275-285 ◽  
Author(s):  
H. Jenkins ◽  
T. Holman ◽  
C. Lyon ◽  
B. Lane ◽  
R. Stick ◽  
...  
Keyword(s):  
Dna Replication ◽  
Nuclear Matrix ◽  
Magnetic Beads ◽  
Major Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Protein Components

Xenopus egg extracts, which support nuclear assembly and DNA replication in vitro, were physically depleted of lamin B3 using monoclonal antibodies linked to magnetic beads. Depleted extracts were still able to support nuclear envelope assembly around demembranated sperm heads but the resulting pronuclei lacked a lamina and were unable to initiate semiconservative DNA replication or to assemble replicases, confirming previous data. Immunoblotting analysis of isolated nuclei and nuclear matrix fractions indicated that lamin-depleted nuclei still accumulated nucleoporins and PCNA. Furthermore, the rate of PCNA uptake was identical in lamin-depleted and control nuclei. However, neither the nucleoporins nor the PCNA was associated with nuclear matrix fractions. The major protein components of sperm pronuclear matrix fractions were characterized by two-dimensional gel electrophoresis. Of these proteins only three out of 22 species, other than the lamins, were significantly reduced in lamin-depleted nuclei, indicating that these nuclei do assemble a nuclear matrix.

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.

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.

scholarly journals Mistargeting of B-Type Lamins at the End of Mitosis

2001 ◽  
Vol 153 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Rikke L. Steen ◽  
Philippe Collas
Keyword(s):  
Cell Survival ◽  
Protein Phosphatase ◽  
Nuclear Lamina ◽  
Lymphoid Cells ◽  
Protein Phosphatase 1 ◽  
Rapid Synthesis ◽  
Gene Encoding ◽  
Nuclear Assembly ◽  
Anchoring Protein

We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C.

scholarly journals Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication

1994 ◽  
Vol 107 (12) ◽  
pp. 3259-3269 ◽  
Author(s):  
C.J. Hutchison ◽  
J.M. Bridger ◽  
L.S. Cox ◽  
I.R. Kill
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Nuclear Lamina ◽  
Proliferating Cells ◽  
Intermediate Filament Proteins ◽  
Nuclear Assembly ◽  
Residual Structure ◽  
Clear Cut ◽  
Germinal Vesicles ◽  
Nuclear Envelope Assembly

The major residual structure that remains associated with the nuclear envelope following extraction of isolated nuclei or oocyte germinal vesicles with non-ionic detergents, nucleases and high salt is the lamina (Fawcett, 1966; Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The nuclear lamina is composed of intermediate filament proteins, termed lamins (Gerace and Blobel, 1980; Shelton et al., 1980), which polymerise to form a basket-weave lattice of fibrils, which covers the entire inner surface of the nuclear envelope and interlinks nuclear pores (Aebi et al., 1986; Stewart and Whytock, 1988; Goldberg and Allen, 1992). At mitosis, the nuclear envelope and the lamina both break down to allow chromosome segregation. As a consequence, each structure has to be rebuilt during anaphase and telophase, allowing cells an opportunity to reposition chromosomes (Heslop-Harrison and Bennett, 1990) and to reorganise looped chromatin domains (Franke, 1974; Franke et al., 1981; Hochstrasser et al., 1986), which may in turn control the use of subsets of genes. Because of the position that it occupies, its dynamics during mitosis and the fact that it is an essential component of proliferating cells, the lamina has been assigned a number of putative roles both in nuclear metabolism and in nuclear envelope assembly (Burke and Gerace, 1986; Nigg, 1989). However, to date there is little clear cut evidence that satisfactorily explains the function of the lamina in relation to its structure. In this Commentary we will describe some of the recent work that addresses this problem and attempt to provide a unified model for the role of lamins in nuclear envelope assembly and for the lamina in the initiation of DNA replication.

scholarly journals Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

1997 ◽  
Vol 136 (6) ◽  
pp. 1201-1212 ◽  
Author(s):  
Timothy P. Spann ◽  
Robert D. Moir ◽  
Anne E. Goldman ◽  
Reimer Stick ◽  
Robert D. Goldman
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Nuclear Lamina ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Lamins ◽  
Egg Extracts ◽  
Elongation Phase

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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 Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A.

1989 ◽  
Vol 8 (12) ◽  
pp. 3891-3898 ◽  
Author(s):  
D.M. Virshup ◽  
M.G. Kauffman ◽  
T.J. Kelly
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cellular Protein ◽  
Phosphatase 2A ◽  
Sv40 Dna
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