Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly

1990 ◽  
Vol 95 (3) ◽  
pp. 383-391
Author(s):  
J.J. Blow ◽  
A.M. Sleeman
Keyword(s):  
Dna Replication ◽  
Interphase Nuclei ◽  
Nuclear Assembly ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Initiation Of Replication ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication ◽  
High Concentrations ◽  
Very High

Purified DNA undergoes a single round of semiconservative replication when incubated in extracts of Xenopus eggs. These extracts also assemble purified DNA into pseudo-nuclei, structures closely resembling normal interphase nuclei. In this paper we show that although less than 60% of purified DNA is assembled into pseudo-nuclei, DNA replication takes place only within these pseudo-nuclei. Further, when nuclear assembly is prevented, the initiation of replication on purified DNA molecules does not occur. In contrast to previous reports, we show that the initiation of DNA replication occurs only during interphase and not during mitosis, even when very high concentrations of purified DNA are used. These experiments show that nuclear formation is a general requirement for the initiation of DNA replication in this system.

Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract

1997 ◽  
Vol 110 (21) ◽  
pp. 2745-2758 ◽  
Author(s):  
Z.H. Lu ◽  
D.B. Sittman ◽  
D.T. Brown ◽  
R. Munshi ◽  
G.H. Leno
Keyword(s):  
Dna Replication ◽  
Nuclear Lamina ◽  
Sperm Chromatin ◽  
Linker Histones ◽  
Nuclear Assembly ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Initiation Of Replication ◽  
Xenopus Egg ◽  
Assembly Analysis

We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.

scholarly journals In vitro assembly of prenucleolar bodies in Xenopus egg extract.

1992 ◽  
Vol 118 (6) ◽  
pp. 1297-1304 ◽  
Author(s):  
P Bell ◽  
M C Dabauvalle ◽  
U Scheer
Keyword(s):  
Experimental System ◽  
Nucleolus Organizer ◽  
Structural Element ◽  
Interphase Nuclei ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Functional Features ◽  
Nucleolar Components

Nuclei assembled in Xenopus egg extract from purified DNA or chromatin resemble their natural counterparts in a number of structural and functional features. However, the most obvious structural element of normal interphase nuclei, the nucleolus, is absent from the in vitro reconstituted nuclei. By EM, cytological silver staining, and immunofluorescence microscopy employing antibodies directed against various nucleolar components we show that nuclei assembled in vitro contain numerous distinct aggregates that resemble prenucleolar bodies (PNBs) by several criteria. Formation of these PNB-like structures requires pore complex-mediated nuclear transport of proteins but is independent of the genetic content of the in vitro nuclei as well as transcriptional and translational events. Our data indicate that nuclei assembled in vitro are capable of initiating early steps of nucleologenesis but that the resulting PNBs are unable to fuse with each other, probably due to the absence of a functional nucleolus organizer. With appropriate modifications, this experimental system should be useful to define and analyze conditions promoting the site-specific assembly of PNBs into a coherent nucleolar body.

scholarly journals Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

1995 ◽  
Vol 15 (6) ◽  
pp. 2942-2954 ◽  
Author(s):  
D M Gilbert ◽  
H Miyazawa ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
Nuclear Membrane ◽  
S Phase ◽  
G1 Phase ◽  
Site Specific ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

scholarly journals Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor

1993 ◽  
Vol 122 (5) ◽  
pp. 985-992 ◽  
Author(s):  
D Coverley ◽  
CS Downes ◽  
P Romanowski ◽  
RA Laskey
Keyword(s):  
Dna Replication ◽  
Nuclear Membrane ◽  
Direct Evidence ◽  
Membrane Permeabilization ◽  
Membrane Repair ◽  
Xenopus Egg Extract ◽  
Nuclear Membranes ◽  
Egg Extract ◽  
Licensing Factor ◽  
Xenopus Egg

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.

scholarly journals Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract

2002 ◽  
Vol 115 (15) ◽  
pp. 3159-3169 ◽  
Author(s):  
Takayuki Kobayashi ◽  
Shusuke Tada ◽  
Takashi Tsuyama ◽  
Hiromu Murofushi ◽  
Masayuki Seki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Focus Formation ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI,discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.

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