DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure.
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 differentiated Xenopus 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 H1(0) 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.
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Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
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D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D064168:Minichromosome Maintenance Complex Component 7; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D051738:Origin Recognition Complex; D010063:Ovum; D014981:Xenopus; D029867:Xenopus Proteins
C118388:Cdc6 protein, Xenopus; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; C102178:XOrc1 protein, Xenopus; D029867:Xenopus Proteins; C509836:Mcm7 protein, Xenopus; C491086:Mcm7 protein, mouse; D064168:Minichromosome Maintenance Complex Component 7