DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function

The lectin, wheat germ agglutinin (WGA), has previously been shown to prevent transport into the cell nucleus. This paper shows that WGA also inhibits nuclear DNA replication, under the same conditions that prevent transport. Although WGA eliminates sperm nuclear DNA replication in a cell-free extract of Xenopus eggs, DNA synthesis on a single-stranded template proceeds normally. Inhibition of nuclear DNA replication is partially reversed by addition of N-acetylglucosamine, and completely reversed by triacetylchitotriose. Sensitivity to inhibition by WGA is greatest during the nuclear assembly phase, and nuclear formation on sperm chromatin is blocked. DNA replication in preformed nuclear templates is also sensitive to WGA inhibition. I propose that WGA blocks DNA replication by preventing nuclear transport. The data presented here also indicate that, under certain circumstances, the elongation stage of DNA replication does not proceed in the absence of an intact nuclear envelope. The roles of the nuclear envelope and active nuclear transport in DNA replication are discussed.

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Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.

The Journal of Cell Biology ◽

10.1083/jcb.106.1.1 ◽

1988 ◽

Vol 106 (1) ◽

pp. 1-12 ◽

Cited By ~ 122

Author(s):

M A Sheehan ◽

A D Mills ◽

A M Sleeman ◽

R A Laskey ◽

J J Blow

Keyword(s):

Dna Replication ◽

Soluble Fraction ◽

Replication Initiation ◽

Sperm Chromatin ◽

Nuclear Pore ◽

Free System ◽

Cell Free System ◽

Nuclear Assembly ◽

Single Membrane ◽

A Cell

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.

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

The Journal of Cell Biology ◽

10.1083/jcb.125.4.705 ◽

1994 ◽

Vol 125 (4) ◽

pp. 705-719 ◽

Cited By ~ 77

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.8.10.1955 ◽

1997 ◽

Vol 8 (10) ◽

pp. 1955-1970 ◽

Cited By ~ 29

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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Synthesis of Sendai Virus Polypeptides by a Cell-free Extract from Wheat Germ

Journal of General Virology ◽

10.1099/0022-1317-33-1-117 ◽

1976 ◽

Vol 33 (1) ◽

pp. 117-123 ◽

Cited By ~ 3

Author(s):

J. W. Davies ◽

A. Portner ◽

D. W. Kingsbury

Keyword(s):

Wheat Germ ◽

Sendai Virus ◽

Cell Free Extract ◽

A Cell

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A lamin-independent pathway for nuclear envelope assembly.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2247 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2247-2259 ◽

Cited By ~ 199

Author(s):

J W Newport ◽

K L Wilson ◽

W G Dunphy

Keyword(s):

Nuclear Envelope ◽

Dna Synthesis ◽

Structural Integrity ◽

Nuclear Lamina ◽

Limited Extent ◽

Nuclear Pores ◽

Nuclear Assembly ◽

Nuclear Envelope Assembly ◽

A Cell ◽

Nuclear Envelope Formation

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.

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Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs

Cell ◽

10.1016/0092-8674(86)90622-7 ◽

1986 ◽

Vol 47 (4) ◽

pp. 577-587 ◽

Cited By ~ 340

Author(s):

J.Julian Blow ◽

Ronald A. Laskey

Keyword(s):

Dna Replication ◽

Cell Free Extract ◽

A Cell ◽

Initiation Of Dna Replication

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Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication

Journal of Cell Science ◽

10.1242/jcs.97.1.177 ◽

1990 ◽

Vol 97 (1) ◽

pp. 177-184

Author(s):

L.S. Cox ◽

G.H. Leno

Keyword(s):

Dna Replication ◽

Xenopus Laevis ◽

High Speed ◽

De Novo ◽

Soluble Fraction ◽

Sperm Chromatin ◽

Nuclear Assembly ◽

Egg Extract ◽

Egg Extracts ◽

Sperm Nuclei

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

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Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability

PROTOPLASMA ◽

10.1007/s00709-015-0861-y ◽

2015 ◽

Vol 253 (3) ◽

pp. 943-956 ◽

Cited By ~ 4

Author(s):

Magda Dubińska-Magiera ◽

Magdalena Chmielewska ◽

Katarzyna Kozioł ◽

Magdalena Machowska ◽

Christopher J. Hutchison ◽

...

Keyword(s):

Cell Death ◽

Protein Expression ◽

Nuclear Envelope ◽

Cell Viability ◽

Cell Nucleus ◽

Chromatin Structure ◽

Integral Membrane Proteins ◽

Nuclear Abnormalities ◽

Lamin B ◽

A Cell

Abstract Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

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