scholarly journals DNA Replication in Quiescent Cell Nuclei: Regulation by the Nuclear Envelope and Chromatin Structure

1999 ◽  
Vol 10 (12) ◽  
pp. 4091-4106 ◽  
Author(s):  
Zhi Hong Lu ◽  
Hongzhi Xu ◽  
Gregory H. Leno
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Cell Nuclei ◽  
Free System ◽  
Replication Complex ◽  
Linker Histones ◽  
Egg Extract ◽  
Initiation Of Replication ◽  
Stable Association

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiatedXenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H10 are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.

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 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.

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.

scholarly journals Remodeler Catalyzed Nucleosome Repositioning: Influence of Structure and Stability

2020 ◽  
Vol 22 (1) ◽  
pp. 76
Author(s):  
Aaron Morgan ◽  
Sarah LeGresley ◽  
Christopher Fischer
Keyword(s):  
Free Energy ◽  
Dna Replication ◽  
Recent Work ◽  
Chromatin Remodeling ◽  
Chromatin Structure ◽  
Genetic Information ◽  
Molecular Machines ◽  
Mechanical Work ◽  
Eukaryotic Genome ◽  
Hydrolysis Of

The packaging of the eukaryotic genome into chromatin regulates the storage of genetic information, including the access of the cell’s DNA metabolism machinery. Indeed, since the processes of DNA replication, translation, and repair require access to the underlying DNA, several mechanisms, both active and passive, have evolved by which chromatin structure can be regulated and modified. One mechanism relies upon the function of chromatin remodeling enzymes which couple the free energy obtained from the binding and hydrolysis of ATP to the mechanical work of repositioning and rearranging nucleosomes. Here, we review recent work on the nucleosome mobilization activity of this essential family of molecular machines.

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