scholarly journals Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei.

1996 ◽  
Vol 133 (5) ◽  
pp. 955-969 ◽  
Author(s):  
J Fang ◽  
R M Benbow
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Nuclear Dna ◽  
Cultured Cells ◽  
Sperm Chromatin ◽  
Quiescent State ◽  
Cell Nuclei ◽  
A6 Cells ◽  
Egg Extracts ◽  
Liver Nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.

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

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.

scholarly journals RCC1, a regulator of mitosis, is essential for DNA replication.

1992 ◽  
Vol 12 (8) ◽  
pp. 3337-3345 ◽  
Author(s):  
M Dasso ◽  
H Nishitani ◽  
S Kornbluth ◽  
T Nishimoto ◽  
J W Newport
Keyword(s):  
Dna Replication ◽  
Regulatory System ◽  
Sperm Chromatin ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Replication Complex ◽  
Temperature Sensitive Mutants ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

scholarly journals The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts.

1991 ◽  
Vol 112 (4) ◽  
pp. 557-566 ◽  
Author(s):  
G H Leno ◽  
R A Laskey
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Fluorescent Intensity ◽  
Free System ◽  
Egg Extract ◽  
Individual Unit ◽  
Egg Extracts ◽  
Replication Foci

We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.

scholarly journals Cadmium (II)-Induced Oxidative Stress Results in Replication Stress and Epigenetic Modifications in Root Meristem Cell Nuclei of Vicia faba

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 640
Author(s):  
Aneta Żabka ◽  
Konrad Winnicki ◽  
Justyna Teresa Polit ◽  
Mateusz Wróblewski ◽  
Janusz Maszewski
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Dna ◽  
Chromatin Condensation ◽  
Replication Stress ◽  
Root Apical Meristem ◽  
H2o2 Production ◽  
Cell Nuclei ◽  
Meristem Cell

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

Differential replication capacities of G1 and S-phase extracts from sea urchin eggs

1993 ◽  
Vol 104 (2) ◽  
pp. 565-572
Author(s):  
H. Zhang ◽  
J.V. Ruderman
Keyword(s):  
Dna Synthesis ◽  
Sea Urchin ◽  
Nuclear Dna ◽  
Specific Pattern ◽  
S Phase ◽  
Sperm Chromatin ◽  
G1 Arrest ◽  
Sea Urchin Eggs ◽  
Egg Extracts ◽  
Fertilized Egg

Sea urchin eggs are arrested in G1 of the first mitotic cell cycle. Fertilization triggers release from G1 arrest and the onset of DNA synthesis about 20 minutes later, even when protein synthesis is blocked. Here we describe extracts from eggs and S-phase embryos that reproduce this stage-specific pattern of DNA synthesis. Fertilized egg extracts formed nuclear membranes around decondensed Xenopus sperm chromatin whereas unfertilized egg extracts did not. Aphidicolin-sensitive deoxynucleotide incorporation was high in extracts of fertilized S-phase eggs and low in those of unfertilized eggs. In contrast, single-stranded DNA templates directed high rates of incorporation in both unfertilized and fertilized egg extracts, suggesting that the stage-specific activities in nuclear DNA synthesis is restricted to initiation on double-stranded DNA. Mixing experiments showed that unfertilized eggs do not contain a dominant inhibitor of replication, nor does fertilization induce the appearance of a soluble, dominant activator.

scholarly journals Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly.

1994 ◽  
Vol 126 (3) ◽  
pp. 591-601 ◽  
Author(s):  
S Dimitrov ◽  
M C Dasso ◽  
A P Wolffe
Keyword(s):  
Xenopus Laevis ◽  
Sperm Nucleus ◽  
Linker Histone ◽  
Core Histone ◽  
Sperm Chromatin ◽  
Histone Phosphorylation ◽  
Egg Extracts ◽  
Core Histones ◽  
Histone Octamers

We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions.

scholarly journals pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

1998 ◽  
Vol 143 (6) ◽  
pp. 1437-1446 ◽  
Author(s):  
Fabien Cubizolles ◽  
Vincent Legagneux ◽  
René Le Guellec ◽  
Isabelle Chartrain ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Cultured Cells ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Maternal Mrna ◽  
Early Embryos ◽  
Egg Extracts ◽  
Cytostatic Factor ◽  
Cell Expression ◽  
Mitotic Chromosome Condensation

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

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

1992 ◽  
Vol 101 (1) ◽  
pp. 43-53 ◽  
Author(s):  
L.S. Cox
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Cell Nucleus ◽  
Wheat Germ ◽  
Nuclear Dna ◽  
Nuclear Transport ◽  
Sperm Chromatin ◽  
Cell Free Extract ◽  
Nuclear Assembly ◽  
A Cell

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.

Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication

1996 ◽  
Vol 109 (2) ◽  
pp. 309-318 ◽  
Author(s):  
T. Krude ◽  
C. Musahl ◽  
R.A. Laskey ◽  
R. Knippers
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Replication Proteins ◽  
Cell Nuclei ◽  
Single Cycle ◽  
Interphase Cells ◽  
Egg Extracts ◽  
Replication Foci ◽  
Mcm Proteins ◽  
Two Populations

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.

RCC1, a regulator of mitosis, is essential for DNA replication

1992 ◽  
Vol 12 (8) ◽  
pp. 3337-3345
Author(s):  
M Dasso ◽  
H Nishitani ◽  
S Kornbluth ◽  
T Nishimoto ◽  
J W Newport
Keyword(s):  
Dna Replication ◽  
Regulatory System ◽  
Sperm Chromatin ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Replication Complex ◽  
Temperature Sensitive Mutants ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

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